Compounds that inhibit the interaction between signal-transducing proteins and the glgf (pdz/dhr) domain and uses thereof

ABSTRACT

This invention provides for a composition capable of inhibiting specific binding between a signal-transducing protein and a cytoplasmic protein. This invention also provides a method of identifying a compound capable of inhibiting specific binding between a signal-transducing protein and a cytoplasmic protein. This invention also provides a method of inhibiting the proliferation of cancer cells. This invention also provides a method of treating cancer with a composition in an amount effective to result in an amount in apoptosis of the cells. This invention also provides a method of inhibiting the proliferation of virally infected cells. This invention also provides for a method of treating a virally-infected subject with a composition in an amount effective to result in apoptosis of the cells. This invention also provides for pharmaceutical compositions.

[0001] The invention disclosed herein was made with Government supportunder Grant No. R01GM55147-01 from the National Institutes of Health ofthe United States Department of Health and Human Services. Accordingly,the U.S. Government has certain rights in this invention.

BACKGROUND

[0002] Throughout this application, various publications are referencedby author and date. Full citations for these publications may be foundlisted alphabetically at the end of the specification immediatelypreceding Sequence Listing and the claims. The disclosures of thesepublications in their entireties are hereby incorporated by referenceinto this application in order to more fully describe the state of theart as known to those skilled therein as of the date of the inventiondescribed and claimed herein.

[0003] Fas (APO-1/CD95) and its ligand have been identified as importantsignal-mediators of apoptosis (Itoh, et al. 1991) The structuralorganization of Fas (APO-1/CD95) has suggested that it is a member ofthe tumor necrosis factor receptor superfamily, which also includes thep75 nerve growth factor receptor (NGFR) (Johnson, et al. 1986), theT-cell-activation marker CD27 (Camerini, et al. 1991), theHodgkin-lymphoma-associated antigen CD30 (Smith, et al. (1993), thehuman B cell antigen CD40 (Stamenkovic, et al. 1989), and T cell antigenOX40 (Mallett, et al. 1990). Genetic mutations of both Fas and itsligand have been associated with lymphoproliferative and autoimmunedisorders in mice (Watanabe-Fukunaga, et al. 1992; Takahashi, et al.1994).

[0004] Furthermore, alterations of Fas expression level have beenthought to lead to the induction of apoptosis in T-cells infected withhuman immunodeficiency virus (HIV) (Westendorp, et al. 1995).

[0005] Several Fas-interacting signal transducing molecules, such asFas-associated phosphatase-1 (FAP-1) (FIG. 1) (Sato, et al. 1995)FADD/MORT1/CAP-1/CAP-2 (Chinnaiyan, et al. 1995; Boldin, et al. 1995;Kischkel, et al. 1995) and RIP (Stanger, et al. 1995), have beenidentified using yeast two-hybrid and biochemical approaches. All butFAP-1 associate with the functional cell death domain of Fas andoverexpression of FADD/MORT1 or RIP induces apoptosis in cellstransfected with these proteins. In contrast, FAP-1 is the only proteinthat associates with the negative regulatory domain (C-terminal 15 aminoacids) (Ito, et al. 1993) of Fas and that inhibits Fas-inducedapoptosis.

[0006] FAP-1 (PTPN13) has several alternatively-spliced forms that areidentical to PTP-BAS/hPTP1E/PTPL1, (Maekawa, et al. 1994; Banville, etal. 1994; Saras, et al. 1994) and contains a membrane-binding regionsimilar to those found in the cytoskeleton-associated proteins, ezrin,(Gould et al. 1989) radixin (Funayama et al. 1991) moesin (Lankes, etal. 1991), neurofibromatosis type II gene product (NFII) (Rouleau, etal. 1993), and protein 4.1 (Conboy, et al. 1991), as well as in thePTPases PTPH1 (Yang, et al. 1991), PTP-MEG (Gu, et al. 1991), and PTPD1(Vogel, et al. 1993). FAP-1 intriguingly contains six GLGF (PDZ/DHR)repeats that are thought to mediate intra-and inter-molecularinteractions among protein domains. The third GLGF repeat of FAP-1 wasfirst identified as a domain showing the specific interaction with theC-terminus of Fas receptor (Sato, et al. 1995). This suggests that theGLGF domain may play an important role in targeting proteins to thesubmembranous cytoskeleton and/or in regulating biochemical activity.GLGF repeats have been previously found in guanylate kinases, as well asin the rat post-synaptic density protein (PSD-95) (Cho, et al. 1992),which is a homolog of the Drosophila tumor suppressor protein,lethal-(1)-disc-large-1 [dlg-1] (Woods, et al 1991; Kitamura, et al.1994). These repeats may mediate homo- and hetero-dimerization, whichcould potentially influence PTPase activity, binding to Fas, and/orinteractions of FAP-1 with other signal transduction proteins. Recently,it has also been reported that the different PDZ domains of proteinsinteract with the C-terminus of ion channels and other proteins (FIG. 1)(TABLE 1)(Kornau, et al. 1995; Kim, et al. 1995; Matsumine, et al.1996). TABLE 1 Proteins that interact with PDZ domains. C-terminalAssociated Protein sequence protein Reference Fas (APO-1/CD95) SLV FAP-12 NMDA receptor SDV PSD95 3 NR2 subunit Shaker-type K+ TDV PSD95 & DLG 4channel APC TEV DLG 5

SUMMARY OF THE INVENTION

[0007] This invention provides a composition capable of inhibitingspecific binding between a signal-transducing protein and a cytoplasmicprotein containing the amino acid sequence (G/S/A/E)-L-G-(F/I/L)(Sequence I.D. No. 1). Further, the cytoplasmic protein may contain theamino acid sequence (K/R/Q)-X_(n)-(G/S/A/E)-L-G-(F/I/L) (Sequence I.D.No. 2), wherein X represents any amino acid which is selected from thegroup comprising the twenty naturally occurring amino acids and nrepresents at least 2, but not more than 4. In a preferred embodiment,the amino acid sequence is SLGI (Sequence I.D. No. 3). Further, theinvention provides for a composition when the signal-transducing proteinhas at its carboxyl terminus the amino acid sequence (S/T)-X-(V/I/L)(Sequence I.D. No. 4), wherein each—represents a peptide bond, eachparenthesis encloses amino acids which are alternatives to one other,each slash within such parentheses separating the alternative aminoacids, and the X represents any amino acid which is selected from thegroup comprising the twenty naturally occurring amino acids.

[0008] This invention also provides for a method of identifying acompound capable of inhibiting specific binding between asignal-transducing protein and a cytoplasmic protein containing theamino acid sequence (G/S/A/E)-L-G-(F/I/L). Further this inventionprovides for a method of identifying a compound capable of inhibitingspecific binding between a signal-transducing protein having at itscarboxyl terminus the amino acid sequence (S/T)-X-(V/L/I) and acytoplasmic protein.

[0009] This invention also provides for a method inhibiting theproliferation of cancer cells, specifically, where the cancer cells arederived from organs comprising the colon, liver, breast, ovary, testis,lung, stomach, spleen, kidney, prostate, uterus, skin, head, thymus andneck, or the cells are derived from either T-cells or B-cells.

[0010] This invention also provides for a method of treating cancer in asubject in an amount of the composition of effective to result inapoptosis of the cells, specifically, where the cancer cells are derivedfrom organs comprising the thymus, colon, liver, breast, ovary, testis,lung, stomach, spleen, kidney, prostate, uterus, skin, head and neck, orthe cells are derived from either T-cells or B-cells.

[0011] This invention also provides for a method of inhibiting theproliferation of virally infected cells, specifically wherein thevirally infected cells are infected with the Hepatitis B virus,Epstein-Barr virus, influenza virus, Papilloma virus, Adenovirus, HumanT-cell lymphtropic virus, type 1 or HIV.

[0012] This invention also provides a pharmaceutical compositioncomprising compositions capable of inhibiting specific binding between asignal-transducing protein and a cytoplasmic protein.

[0013] This invention also provides a pharmaceutical compositioncomprising compounds identified to be capable of inhibiting specificbinding between a signal-transducing protein and a cytoplasmic protein.

BRIEF DESCRIPTION OF THE FIGURES

[0014]FIG. 1. Diagram of Fas-associated phosphatase-1 protein, showingthe six GLGF (PDZ/DHR) domain repeats; comparison of similar membranebinding sites with other proteins and proteins that contain GLGF(PDZ/DHR) repeats.

[0015]FIGS. 2A, 2B, 2C and 2D. Mapping of the minimal region of theC-terminal of Fas required for the binding to FAP-1. Numbers at rightshow each independent clone (FIGS. 2C and 2D).

[0016]2A. Strategy for screening of a random peptide library by theyeast two-hybrid system.

[0017]2E. Alignment of the C-terminal 15 amino acids of Fas betweenhuman (Sequence I.D. No. 5), rat (Sequence I.D. No. 6), and mouse(Sequence I.D. No. 7).

[0018]2C. The results of screening a semi-random peptide library. Toprow indicates the amino acids which were fixed based on the homologybetween human and rat. Dash lines show unchanged amino acids.

[0019]2D. The results of screening a random peptide library (SequenceI.D. No. 8, Sequence I.D. No. 9, Sequence I.D. No. 10, Sequence I.D. No.11, Sequence I.D. No. 12, Sequence I.D. No. 13, Sequence I.D. No. 14,Sequence I.D. No. 15, Sequence I.D. No. 16, Sequence I.D. No. 17,respectively).

[0020]FIGS. 3A, 3B and 3C. Inhibition assay of Fas/FAP-1 binding invitro.

[0021]3A. Inhibition assay of Fas/FAP-1 binding using the C-terminal 15amino acids of Fas. GST-Fas fusion protein (191-355) was used for invitro binding assay (lane 1, 3-10). GST-Fas fusion protein (191-320)(lane 2) and 1 mM human PAMP (N-terminal 20 amino acids ofproadrenomedullin, M.W. 2460.9) (lane 3) were used as negative controls.The concentrations of the C-terminal 15 amino acids added were 1 (lane4), 3 (lane 5), 10 (lane 6), 30 (lane 7), 100 (lane 8), 300 (lane 9),and 1000 μM (lane 10).

[0022]3B. Inhibition assay of Fas/FAP-1 binding using the truncatedpeptides corresponding to the C-terminal 15 amino acids of Fas. Allsynthetic peptides were acetylated for this inhibition assay (SequenceI.D. No. 4, Sequence I.D. No. 18, Sequence I.D. No.: 19, Sequence I.D.No. 20, Sequence I.D. No. 21, Sequence I.D. No. 22, Sequence I.D. No.23, respectively)

[0023]3C. Inhibitory effect of Fas/FAP-1 binding using the scannedtripeptides.

[0024]FIGS. 4A, 4B, 4C and 4D.

[0025]4A. Interaction of the C-terminal 3 amino acids of Fas with FAP-1in yeast.

[0026]4B. Interaction of the C-terminal 3 amino acids of Fas with FAP-1in vitro.

[0027]4C. Immuno-precipitation of native Fas with GST-FAP-1.

[0028]4D. Inhibition of Fas/FAP-1 binding with Ac-SLV or Ac-SLY.

[0029]FIGS. 5A, 5B, 5C, 5D, 5E and 5F. Microinjection of Ac-SLV into theDLD-1 cell line. Triangles identify the cells both that were could bemicroinjected with Ac-SLV and that showed condensed chromatinidentified. On the other hand, only one cell of the area appearedapoptotic when microinjected with Ac-SLY.

[0030]5A. Representative examples of the cells microinjected with Ac-SLVin the presence of 500 ng/ml CH11 are shown in phase contrast.

[0031]5B. Representative examples of the cells microinjected with AC-SLYin the presence of 500 ng/ml CH11 are shown in phase contrast.

[0032]5C. Representative examples of the cells microinjected with Ac-SLVin the presence of 500 ng/ml CH11 are shown stained with FITC.

[0033]5D. Representative examples of the cells microinjected with AC-SLYin the presence of 500 ng/ml CH11 are shown stained with FITC.

[0034]5E. Representative examples of the cells microinjected with Ac-SLVin the presence of 500 ng/ml CH11 are shown with fluorescent DNAstaining with Hoechst 33342.

[0035]5F. Representative examples of the cells microinjected with AC-SLYin the presence of 500 ng/ml CH11 are shown in fluorescent DNA stainingwith Hoechst 33342.

[0036]FIG. 6. Quantitation of apoptosis in microinjected DLD-1 cells.

[0037]FIGS. 7A, 7B, 7C, 7D, 7E, 7F, 7G, and 7H.

[0038]7A. Amino acid sequence of human nerve growth factor receptor(Sequence I.D. No. 24).

[0039]7B. Amino acid sequence of human CD4 receptor (Sequence I.D. No.25).

[0040]7C. The interaction of Fas-associated phosphatase-1 to theC-terminal of nerve growth factor receptor (NGFR)(p75).

[0041]7D. Amino acid sequence of human colorectal mutant cancer protein(Sequence I.D. No. 26).

[0042]7E. Amino acid sequence of protein kinase C, alpha type.

[0043]7F. Amino acid sequence of serotonin 2A receptor (Sequence I.D.No. 27).

[0044]7G. Amino acid sequence of serotonin 2B receptor (Sequence I.D.No. 28).

[0045]7H. Amino acid sequence of adenomatosis polyposis coli protein(Sequence I.D. No. 29).

[0046]FIG. 8. Representation of the structural characteristics of p75NGFR (low-affinity nerve growth factor receptor).

[0047]FIG. 9. Comparison of the C-terminal ends of Fas and p75 NGFR.

[0048]FIG. 10. In vitro interaction of ³⁵S-labeled FAP-1 with variousreceptors expressed as GST fusion proteins. The indicated GST fusionproteins immobilized on glutathione-Sepharose beads were incubated within vitro translated, ³⁵S-labeled FAP-1 protein. After the beads werewashed, retained FAP-1 protein was analyzed by SDS-PAGE andautoradiography.

[0049]FIGS. 11A and 11B. In vitro interaction ³⁵S-labeled FAP-1 withGST-p75 deletion mutants.

[0050]11A. Schematic representation of the GST fusion proteinscontaining the cytoplasmic domains of p75 and p75 deletion mutants.Binding of FAP-1 to the GST fusion proteins with various p75 deletionmutants is depicted at the right and is based on data from (11B).

[0051]11B. Interaction of in vitro translated, ³⁵S-labeled FAP-1 proteinwith various GST fusion proteins immobilized on glutathione-Sepharosebeads. After the beads were washed, retained FAP-1 protein was analyzedby SDS-PAGE and autoradiography.

[0052]FIG. 12. The association between LexA-C-terminal cytoplasmicregion of p75NGFR and VP16-FAP-1. The indicated yeast strains wereconstructed by transformation and the growth of colonies was tested. +/−indicates the growth of colonies on his ⁻ plate.

DETAILED DESCRIPTION OF THE INVENTION

[0053] As used herein, amino acid residues are abbreviated as follows:A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys;L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val;W, Trp; and Y, Tyr.

[0054] In order to facilitate an understanding of the material whichfollows, certain frequently occurring methods and/or terms are bestdescribed in Sambrook, et al., 1989.

[0055] The present invention provides for a composition capable ofinhibiting specific binding between a signal-transducing protein and acytoplasmic protein containing the amino acid sequence(G/S/A/E)-L-G-(F/I/L), wherein each—represents a peptide bond, eachparenthesis encloses amino acids which are alternatives to one other,and each slash within such parentheses separating the alternative aminoacids. Further, the cytoplasmic protein may contain the amino acidsequence (K/R/Q)-X_(n)-(G/S/A/E)-L-G-(F/I/L), wherein X represents anyamino acid which is selected from the group comprising the twentynaturally occurring amino acids and n represents at least 2, but notmore than 4. Specifically, in a preferred embodiment, the cytoplasmicprotein contains the amino acid sequence SLGI.

[0056] The amino acid sequence (K/R/Q)-X_(n)-(G/S/A/E)-L-G-(F/I/L) isalso well-known in the art as “GLGF (PDZ/DHR) amino acid domain.” Asused herein, “GLGF (PDZ/DHR) amino acid domain” means the amino acidsequence (K/R/Q)-X_(n)-(G/S/A/E)-L-G-(F/I/L).

[0057] In a preferred embodiment, the signal-transducing protein has atits carboxyl terminus the amino acid sequence (S/T)-X-(V/I/L), whereineach—represents a peptide bond, each parenthesis encloses amino acidswhich are alternatives to one other, each slash within such parenthesesseparating the alternative amino acids, and the X represents any aminoacid which is selected from the group comprising the twenty naturallyoccurring amino acids.

[0058] The compositions of the subject invention may be, but not limitedto, antibodies, inorganic compounds, organic compounds, peptides,peptidomimetic compounds, polypeptides or proteins, fragments orderivatives which share some or all properties, e.g. fusion proteins.The composition may be naturally occurring and obtained by purification,or may be non-naturally occurring and obtained by synthesis.

[0059] Specifically, the composition may be a peptide containing thesequence (S/T)-X-(V/I/L)-COOH, wherein each—represents a peptide bond,each parenthesis encloses amino acids which are alternatives to oneother, each slash within such parentheses separating the alternativeamino acids, the X represents any amino acid which is selected from thegroup comprising the twenty naturally occurring amino acids. Inpreferred embodiments, the peptide contains one of the followingsequences: DSENSNFRNEIQSLV, RNEIQSLV, NEIQSLV, EIQSLV, IQSLV, QSLV, SLV,IPPDSEDGNEEQSLV, DSEMYNFRSQLASVV, IDLASEFLFLSNSFL, PPTCSQANSGRISTL,SDSNMNMNELSEV, QNFRTYIVSFV, RETIESTV, RGFISSLV, TIQSVI, ESLV. A furtherpreferred embodiment would be an organic compound which has the sequenceAc-SLV-COOH, wherein the Ac represents an acetyl and each—represents apeptide bond.

[0060] An example of the subject invention is provided infra. Acetylatedpeptides may be automatically synthesized on an Advanced ChemTech ACT357using previously published procedures by analogy. Wang resin was usedfor each run and N^(α)-Fmoc protection was used for all amino acids, andthen 20% piperidine/DMF and coupling was completed using DIC/HOBt andsubsequently HBTU/DIEA. After the last amino acid was coupled, thegrowing peptide on the resin was acetylated with Ac₂O/DMF. Theacetylated peptide was purified by HPLC and characterized by FAB-MS and¹H-NMR.

[0061] Further, one skilled in the art would know how to constructderivatives of the above-described synthetic peptides coupled tonon-acetyl groups, such as amines.

[0062] This invention also provides for a composition capable ofinhibiting specific binding between a signal-transducing protein havingat its carboxyl terminus the amino acid sequence (S/T)-X-(V/I/L),wherein each—represents a peptide bond, each parenthesis encloses aminoacids which are alternatives to one other, each slash within suchparentheses separating the alternative amino acids, the X represents anyamino acid which is selected from the group comprising the twentynaturally occurring amino acids, and a cytoplasmic protein.

[0063] The compositions of the subject invention includes antibodies,inorganic compounds, organic compounds, peptides, peptidomimeticcompounds, polypeptides or proteins, fragments or derivatives whichshare some or all properties, e.g. fusion proteins.

[0064] This invention also provides a method of identifying a compoundcapable of inhibiting specific binding between a signal-transducingprotein and a cytoplasmic protein containing the amino acid sequence(G/S/A/E)-L-G-(F/I/L), wherein each—represents a peptide bond, eachparenthesis encloses amino acids which are alternatives to one other,each slash within such parentheses separating the alternative aminoacids, which comprises (a) contacting the cytoplasmic protein bound tothe signal-transducing protein with a plurality of compounds underconditions permitting binding between a known compound previously shownto be able to displace the signal-transducing protein bound to thecytoplasmic protein and the bound cytoplasmic protein to form a complex;and (b) detecting the displaced signal-transducing protein or thecomplex formed in step (a) wherein the displacement indicates that thecompound is capable of inhibiting specific binding between thesignal-transducing protein and the cytoplasmic protein.

[0065] The inhibition of the specific binding between thesignal-transducing protein and the cytoplasmic protein may affect thetranscription activity of a reporter gene.

[0066] Further, in step (b), the displaced cytoplasmic protein or thecomplex is detected by comparing the transcription activity of areporter gene before and after the contacting with the compound in step(a), where a change of the activity indicates that the specific bindingbetween the signal-transducing protein and the cytoplasmic protein isinhibited and the signal-transducing protein is displaced.

[0067] As used herein, the “transcription activity of a reporter gene”means that the expression level of the reporter gene will be alteredfrom the level observed when the signal-transducing protein and thecytoplasmic protein are bound. One can also identify the compound bydetecting other biological functions dependent on the binding betweenthe signal-transducing protein and the cytoplasmic protein. Examples ofreporter genes are numerous and well-known in the art, including, butnot limited to, histidine resistant genes, ampicillin resistant genes,β-galactosidase gene.

[0068] Further the cytoplasmic protein may be bound to a solid support.Also the compound may be bound to a solid support and comprises anantibody, an inorganic compound, an organic compound, a peptide, apeptidomimetic compound, a polypeptide or a protein.

[0069] An example of the method is provided infra. One can identify acompound capable of inhibiting specific binding between thesignal-transducing protein and the cytoplasmic protein using directmethods of detection such as immuno-precipitation of the cytoplasmicprotein and the compound bound to a detectable marker. Further, onecould use indirect methods of detection that would detect the increaseor decrease in levels of gene expression. As discussed infra, one couldconstruct synthetic peptides fused to a LexA DNA binding domain. Theseconstructs would be transformed into the L40-strain with an appropriatecell line having an appropriate reporter gene. One could then detectwhether inhibition had occurred by detecting the levels of expression ofthe reporter gene. In order to detect the expression levels of thereporter gene, one skilled in the art could employ a variety ofwell-known methods, e.g. two-hybrid systems in yeast, mammals or othercells.

[0070] Further, the contacting of step (a) may be in vitro, in vivo, andspecifically in an appropriate cell, e.g. yeast cell or mammalian cell.Examples of mammalian cells include, but not limited to, the mousefibroblast cell NIH 3T3, CHO cells, HeLa cells, Ltk⁻ cells, Cos cells,etc.

[0071] Other suitable cells include, but are not limited to, prokaryoticor eukaryotic cells, e.g. bacterial cells (including gram positivecells), fungal cells, insect cells, and other animals cells.

[0072] Further, the signal-transducing protein may be a cell surfacereceptor, signal transducer protein, or a tumor suppressor protein.Specifically, the cell surface protein is the Fas receptor and may beexpressed in cells derived from organs including, but not limited to,thymus, liver, kidney, colon, ovary, breast, testis, spleen, lung,stomach, prostate, uterus, skin, head, and neck, or expressed in cellscomprising T-cells and B-cells. In a preferred embodiment, the T-cellsare Jurkat T-cells.

[0073] Further, the cell-surface receptor may be a CD4 receptor, p75receptor, serotonin 2A receptor, or serotonin 2B receptor.

[0074] Further, the signal transducer protein may be ProteinKinase-C-α-type.

[0075] Further, the tumor suppressor protein may be a adenomatosispolyposis coli tumor suppressor protein or colorectal mutant cancerprotein.

[0076] Further, the cytoplasmic protein contains the amino acid sequenceSLGI, specifically Fas-associated phosphatase-1.

[0077] This invention also provides a method of identifying a compoundcapable of inhibiting specific binding between a signal-transducingprotein having at its carboxyl terminus the amino acid sequence(S/T)-X-(V/I/L), wherein each—represents a peptide bond, eachparenthesis encloses amino acids which are alternatives to one other,each slash within such parentheses separating the alternative aminoacids, the X represents any amino acid which is selected from the groupcomprising the twenty naturally occurring amino acids, and a cytoplasmicprotein which comprises (a) contacting the signal-transducing proteinbound to the cytoplasmic protein with a plurality of compounds underconditions permitting binding between a known compound previously shownto be able to displace the cytoplasmic protein bound to thesignal-transducing protein and bound signal-transducing protein to forma complex; and (b) detecting the displaced cytoplasmic protein or thecomplex of step (a), wherein the displacement indicates that thecompound is capable of inhibiting specific binding between thesignal-transducing protein and the cytoplasmic protein. The inhibitionof the specific binding between the signal-transducing protein and thecytoplasmic protein affects the transcription activity of a reportergene. Further, in step (b), the displaced signal-transducing protein orthe complex is detected by comparing the transcription activity of areporter gene before and after the contacting with the compound in step(a), where a change of the activity indicates that the specific bindingbetween the signal-transducing protein and the cytoplasmic protein isinhibited and the cytoplasmic protein is displaced.

[0078] Further, in step (b), the displaced cytoplasmic protein or thecomplex is detected by comparing the transcription activity of areporter gene before and after the contacting with the compound in step(a), where a change of the activity indicates that the specific bindingbetween the signal-transducing protein and the cytoplasmic protein isinhibited and the signal-transducing protein is displaced.

[0079] As used herein, the “transcription activity of a reporter gene”means that the expression level of the reporter gene will be alteredfrom the level observed when the signal-transducing protein and thecytoplasmic protein are bound. One can also identify the compound bydetecting other biological functions dependent on the binding betweenthe signal-transducing protein and the cytoplasmic protein. Examples ofreporter genes are numerous and well-known in the art, including, butnot limited to, histidine resistant genes, ampicillin resistant genes,β-galactosidase gene.

[0080] Further, the cytoplasmic protein may be bound to a solid supportor the compound may be bound to a solid support, comprises an antibody,an inorganic compound, an organic compound, a peptide, a peptidomimeticcompound, a polypeptide or a protein.

[0081] An example of the method is provided infra. One could identify acompound capable of inhibiting specific binding between thesignal-transducing protein and the cytoplasmic protein using directmethods of detection such as immuno-precipitation of the cytoplasmicprotein and the compound bound with a detectable marker. Further, onecould use indirect methods of detection that would detect the increaseor decrease in levels of gene expression. As discussed infra, one couldconstruct synthetic peptides fused to a LexA DNA binding domain. Theseconstructs would be transformed into L40-strain with an appropriate cellline having a reporter gene. One could then detect whether inhibitionhad occurred by detecting the levels of the reporter gene. Differentmethods are also well known in the art, such as employing a yeasttwo-hybrid system to detect the expression of a reporter gene.

[0082] Further the contacting of step (a) can be in vitro or in vivo,specifically in a yeast cell or a mammalian cell. Examples of mammaliancells include, but not limited to, the mouse fibroblast cell NIH 3T3,CHO cells, HeLa cells, Ltk⁻ cells, Cos cells, etc.

[0083] Other suitable cells include, but are not limited to, prokaryoticor eukaryotic cells, e.g. bacterial cells (including gram positivecells), fungal cells, insect cells, and other animals cells.

[0084] Further, the signal-transducing protein is a cell surfacereceptor, signal transducer protein, or a tumor suppressor protein.Specifically, the cell surface protein is the Fas receptor and isexpressed in cells derived from organs comprising thymus, liver, kidney,colon, ovary, breast, testis, spleen, stomach, prostate, uterus, skin,head and neck, or expressed in cells comprising T-cells and B-cells. Ina preferred embodiment, the T-cells are Jurkat T-cells.

[0085] Further, the cell-surface receptor may be a CD4 receptor, p75receptor, serotonin 2A receptor, or serotonin 2B receptor.

[0086] Further, the signal transducer protein may be ProteinKinase-C-α-type.

[0087] Further, the tumor suppressor protein may be a adenomatosispolyposis coli tumor suppressor protein or colorectal mutant cancerprotein.

[0088] Further, the cytoplasmic protein contains the amino acid sequenceSLGI, specifically Fas-associated phosphatase-1.

[0089] This invention also provides a method of inhibiting theproliferation of cancer cells comprising the above-describedcomposition, specifically, wherein the cancer cells are derived fromorgans including, but not limited to, thymus, liver, kidney, colon,ovary, breast, testis, spleen, stomach, prostate, uterus, skin, head andneck, or wherein the cancer cells are derived from cells comprisingT-cells and B-cells.

[0090] This invention also provides a method of inhibiting theproliferation of cancer cells comprising the compound identified by theabove-described method, wherein the cancer cells are derived from organsincluding, but not limited to, thymus, liver, kidney, colon, ovary,breast, testis, spleen, stomach, prostate, uterus, skin, head and neck,or wherein the cancer cells are derived from cells comprising T-cellsand B-cells.

[0091] The invention also provides a method of treating cancer in asubject which comprises introducing to the subject's cancerous cells anamount of the above-described composition effective to result inapoptosis of the cells, wherein the cancer cells are derived from organsincluding, but not limited to, thymus, liver, kidney, colon, ovary,breast, testis, spleen, stomach, prostate, uterus, skin, head and neck,or wherein the cancer cells are derived from cells comprising T-cellsand B-cells.

[0092] As used herein “apoptosis” means programmed cell death of thecell. The mechanisms and effects of programmed cell death differs fromcell lysis. Some observable effects of apoptosis are: DNA fragmentationand disintegration into small membrane-bound fragments called apoptoticbodies.

[0093] Means of detecting whether the composition has been effective toresult in apoptosis of the cells are well-known in the art. One means isby assessing the morphological change of chromatin using either phasecontrast or fluorescence microscopy.

[0094] The invention also provides for a method of inhibiting theproliferation of virally infected cells comprising the above-describedcomposition or the compound identified by the above-described, whereinthe virally infected cells comprise Hepatitis B virus, Epstein-Barrvirus, influenza virus, Papilloma virus, Adeno virus, Human T-celllymphtropic virus, type 1 or HIV.

[0095] The invention also provides a method of treating avirally-infected subject which comprises introducing to the subject'svirally-infected cells the above-described composition effective toresult in apoptosis of the cells or the compound identified by theabove-described method of claim 27 effective to result in apoptosis ofthe cells, wherein the virally infected cells comprise the Hepatitis Bvirus, Epstein-Barr virus, influenza virus, Papilloma virus, Adenovirus, Human T-cell lymphtropic virus, type 1 or HIV.

[0096] Means of detecting whether the composition has been effective toresult in apoptosis of the cells are well-known in the art. One means isby assessing the morphological change of chromatin using either phasecontrast or fluorescence microscopy.

[0097] This invention also provides for a pharmaceutical compositioncomprising the above-described composition of in an effective amount anda pharmaceutically acceptable carrier.

[0098] This invention also provides for a pharmaceutical compositioncomprising the compound identified by the above-described method of inan effective amount and a pharmaceutically acceptable carrier.

[0099] This invention further provides a composition capable ofspecifically binding a signal-transducing protein having at its carboxylterminus the amino acid sequence (S/T)-X-(V/L/I), whereineach—represents a peptide bond, each parenthesis encloses amino acidswhich are alternatives to one other, each slash within such parenthesesseparating the alternative amino acids, and the X represents any aminoacid which is selected from the group comprising the twenty naturallyoccurring amino acids. The composition may contain the amino acidsequence (G/S/A/E)-L-G-(F/I/L), wherein each—represents a peptide bond,each parenthesis encloses amino acids which are alternatives to oneother, and each slash within such parentheses separating the alternativeamino acids. In a preferred embodiment, the composition contains theamino acid sequence (K/R/Q)-X_(n)-(G/S/A/E)-L-G-(F/I/L) wherein Xrepresents any amino acid which is selected from the group comprisingthe twenty naturally occurring amino acids and n represents at least 2,but not more than 4. In another preferred embodiment, the compositioncontains the amino acid sequence SLGI.

[0100] This invention further provides a method for identifyingcompounds capable of binding to a signal-transducing protein having atits carboxyl terminus the amino acid sequence (S/T)-X-(V/L/I), whereineach—represents a peptide bond, each parenthesis encloses amino acidswhich are alternatives to one other, each slash within such parenthesesseparating the alternative amino acids, the X represents any amino acidwhich is selected from the group comprising the twenty naturallyoccurring amino acids, which comprises (a) contacting thesignal-transducing protein with a plurality of compounds underconditions permitting binding between a known compound previously shownto be able to bind to the signal-transducing protein to form a complex;and (b) detecting the complex formed in step (a) so as to identify acompound capable of binding to the signal-transducing protein.Specifically, the identified compound contains the amino acid sequence(G/S/A/E)-L-G-(F/I/L). In a further preferred embodiment, the identifiedcompound contains the amino acid sequence SLGI.

[0101] Further, in the above-described method, the signal-transducingprotein may be bound to a solid support. Also, the compound may be boundto a solid support, and may comprise an antibody, an inorganic compound,an organic compound, a peptide, a peptidomimetic compound, a polypeptideor a protein.

[0102] Further, the signal-transducing protein may be a cell-surfacereceptor or a signal transducer. Specifically, the signal-transducingprotein may be the Fas receptor, CD4 receptor, p75 receptor, serotonin2A receptor, serotonin 2B receptor, or protein kinase-C-α-type.

[0103] This invention also provides a method of restoring negativeregulation of apoptosis in a cell comprising the above-describedcomposition or a compound identified by the above-described method.

[0104] As used herein “restoring negative regulation of apoptosis” meansenabling the cell from proceeding onto programmed cell death.

[0105] For example, cells that have functional Fas receptors andFas-associated phosphatase 1 do not proceed onto programmed cell deathor apoptosis due to the negative regulation of Pas by the phosphatase.However, if Fas-associated phosphatase 1 is unable to bind to thecarboxyl terminus of the Fas receptor ((S/T)-X-(V/L/I) region), e.g.mutation or deletion of at least one of the amino acids in the aminoacid sequence (G/S/A/E)-L-G-(F/I/L), the cell will proceed to apoptosis.By introducing a compound capable of binding to the carboxyl terminus ofthe Fas receptor, one could mimic the effects of a functionalphosphatase and thus restore the negative

[0106] regulation of apoptosis. This invention also provides a method ofpreventing apoptosis in a cell comprising the above-describedcomposition or a compound identified by the above-described method.

[0107] This invention also provides a means of treating pathogenicconditions caused by apoptosis of relevant cells comprising theabove-described composition or the compound identified by theabove-described method.

[0108] This invention is illustrated in the Experimental Details sectionwhich follows. These sections are set forth to aid in an understandingof the invention but are not intended to, and should not be construedto, limit in any way the invention as set forth in the claims whichfollow thereafter.

[0109] First Series of Experiments

[0110] Experimental Details

[0111] Methods and Materials

[0112] 1. Screening a Semi-Random and Random Peptide Library.

[0113] To create numerous mutations in a restricted DNA sequence, PCRmutagenesis with degenerate oligonucleotides was employed according to aprotocol described elsewhere (Hill, et al. 1987). Based on the homologybetween human and rat, two palindromic sequences were designed forconstruction of semi-random library. The two primers used were5′-CGGAATTCNNNNNNNNNAACAGCNNNNNNNNNAATGAANNNCAAAGTCTGNN NTGAGGATCCTCA-3′(Seq. I.D. No. 30) and5′-CGGAATTCGACTCAGAANNNNNNAACTTCAGANNNNNNATCNNNNNNNNNGT CTGAGGATCCTCA-3′(Seq. I.D. No. 31). Briefly, the two primers (each 200 pmol), purifiedby HPLC, were annealed at 70° C. for 5 minutes and cooled at 23° C. for60 minutes. A Klenow fragment (5 U) was used for filling in with a dNTPmix (final concentration, 1 mM per each dNTP) at 23° C. for 60 minutes.The reaction was stopped with 1 μl of 0.5 M EDTA and the DNA waspurified with ethanol precipitation. The resulting double-stranded DNAwas digested with EcoRI and BamHI and re-purified by electrophoresis onnon-denaturing polyacrylamide gels. The double-strand oligonucleotideswere then ligated into the EcoRI-BamHI sites of the pBTM116 plasmid. Theligation mixtures were electroporated into the E. coli XL1-Blue MRF′(Stratagene) for the plasmid library. The large scale transformation wascarried out as previously reported. The plasmid library was transformedinto L40-strain cells (MATa, trp1, leu2, his3, ade2, LYS2:(lexAop)⁴-HIS3, URA3::(lexAop)⁸-lacZ) carrying the plasmid pVP16-31containing a FAP-1 cDNA (Sato, et al. 1995). Clones that formed onhistidine-deficient medium (His⁺) were transferred to plates containing40 μg/ml X-gal to test for a blue reaction product (β-gal⁺) in plate andfilter assays. The clones selected by His⁺ and β-gal⁺ assay were testedfor further analysis. The palindromic oligonucleotide,5′-CGGAATTC-(NNN)₄₋₁₅-TGAGGATCCTCA-3′ (Seq. I.D. No. 32), was used forthe construction of the random peptide library.

[0114] 2. Synthesis of Peptides

[0115] Peptides were automatically synthesized on an Advanced ChemTechACT357 by analogy to published procedures (Schnorrenberg and Gerhardt,1989). Wang resin (0.2-0.3 mmole scale) was used for each run andN′-Fmoc protection was employed for all amino acids. Deprotection wasachieved by treatment with 20% piperidine/DMF and coupling was completedusing DIC/HOBt and subsequent HBTU/DIEA. After the last amino acid wascoupled, the growing peptide on the resin was acetylated with Ac₂O/DMF.The peptide was cleaved from the resin with concomitant removal of allprotecting groups by treating with TFA. The acetylated peptide waspurified by HPLC and characterized by FAB-MS and ¹H-NMR.

[0116] 3. Inhibition Asssay of Fas/FAP-1 Binding Using the C-Terminal 15Amino Acids of Fas.

[0117] HFAP-10 cDNA (Sato, et al. 1995) subcloned into the Bluescriptvector pSK-II (Stratagene) was in vitro-translated from an internalmethionine codon in the presence of ³⁵S-L-methionine using a coupled invitro transcription/translation system (Promega, TNT lysate) and T7 RNApolymerase. The resulting ³⁵S-labeled protein was incubated with GST-Fasfusion proteins that had been immobilized on GST-Sepharose 4B affinitybeads (Pharmacia) in a buffer containing 150 mM NaCl, 50 mM Tris [pH8.0], 5 mM DTT, 2 mM EDTA, 0.1% NP-40, 1 mM PMSF, 50 μg/ml leupeptin, 1mM Benzamidine, and 7 μg/ml pepstatin for 16 hours at 4° C. Afterwashing vigorously 4 times in the same buffer, associated proteins wererecovered with the glutathione-Sepharose beads by centrifugation, elutedinto boiling Laemmli buffer, and analyzed by SDS-PAGE and fluorography.

[0118] 4. Inhibition Assay of Terminal 15 Amino Acids of Fas andInhibitory Effect of Fas/FAP-1 Binding Using Diverse Tripeptides.

[0119] In vitro-translated [³⁵]HFAP-1 was purified with a NAP-5 column(Pharmacia) and incubated with 3 μM of GST-fusion proteins for 16 hoursat 4° C. After washing 4 times in the binding buffer, radioactivityincorporation was determined in a b counter. The percentage of bindinginhibition was calculated as follows: percent inhibition=[radioactivityincorporation using GST-Fas (191-335) with peptides−radioactivityincorporation using GST-Fas (191-320) with peptides]/[radioactivityincorporation using GST-Fas (191-335) without peptides−radioactivityincorporation using GST-Fas (191-320) without peptides]. n=3.

[0120] 5. Interaction of the C-Terminal 3 Amino Acids of Fas with FAP-1in Yeast and in Vitro.

[0121] The bait plasmids, pBTM116 (LexA)-SLV, -PLV, -SLY, and -SLA, wereconstructed and transformed into L40-strain with pVP16-FAP-l or -ras.Six independent clones from each transformants were picked up for theanalysis of growth on histidine-deficient medium. GST-Fas, -SLV, and PLVwere purified with GST-Sepharose 4B affinity beads (Pharmacia). Themethods for in vitro binding are described above.

[0122] 6. Immuno-Precipitation of Native Fas with GST-FAP-1 andInhibition of Fas/FAP-1 Binding with Ac-SLV.

[0123] GST-fusion proteins with or without FAP-1 were incubated withcell extracts from Jurkat T-cells expressing Fas. The bound Fas wasdetected by Western analysis using anti-Fas monoclonal antibody (F22120,Transduction Laboratories). The tripeptides, Ac-SLV and Ac-SLY were usedfor the inhibition assay of Fas/FAP-1 binding.

[0124] 7. Microinjection of Ac-SLV into the DLD-1 Cell Line.

[0125] DLD-1 human colon cancer cells were cultured in RPMI 1640 mediumcontaining 10% FCS. For microinjection, cells were plated on CELLocate(Eppendorf) at 1×10⁵ cells/2 ml in a 35 mm plastic culture dish andgrown for 1 day. Just before microinjection, Fas monoclonal antibodiesCH11 (MBL International) was added at the concentration of 500 ng/ml.All microinjection experiments were performed using an automaticmicroinjection system (Eppendorf transjector 5246, micro-manipulator5171 and Femtotips) (Pantel, et al. 1995). Synthetic tripeptides weresuspended in 0.1% (w/v) FITC-Dextran (Sigma)/K-PBS at the concentrationof 100 mM. The samples were microinjected into the cytoplasmic region ofDLD-1 cells. Sixteen to 20 hours postinjection, the cells were washedwith PBS and stained with 10 μg/ml Hoechst 33342 in PBS. Afterincubation at 37° C. for 30 minutes, the cells were photographed and thecells showing condensed chromatin were counted as apoptotic.

[0126] 8. Quantitation of Apoptosis in Microinjected DLD-1 Cells.

[0127] For each experiment, 25-100 cells were microinjected. Apoptosisof microinjected cells was determined by assessing morphological changesof chromatin using phase contrast and fluorescence microscopy (Wang, etal., 1995; McGahon, et al., 1995). The data are means +/−S.D. for two orthree independent determinations.

[0128] Discussion

[0129] In order to identify the minimal peptide stretch in theC-terminal region of the Fas receptor necessary for FAP-1 binding, an invitro inhibition assay of Fas/FAP-1 binding was used using a series ofsynthetic peptides as well as yeast two-hybrid system peptide libraries(FIG. 2A). First, semi-random libraries (based on the homology betweenhuman and rat Fas) (FIGS. 2B and 2C) of 15 amino acids fused to a LexADNA binding domain were constructed and co-transformed into yeast strainL40 with pVP16-31 (Sato, et al. 1995) that was originally isolated asFAP-1. After the selection of 200 His⁺ colonies from an initial screenof 5.0×10⁶ (Johnson, et al. 1986) transformants, 100 colonies that wereβ-galactosidase positive were picked for further analysis. Sequenceanalysis of the library plasmids encoding the C-terminal 15 amino acidsrevealed that all of the C-termini were either valine, leucine orisoleucine residues. Second, a random library of 4-15 amino acids fusedto a LexA DNA binding domain was constructed and screened according tothis strategy (FIG. 2D). Surprisingly, all of the third amino acidresidues from the C-termini were serine, and the results of C-terminalamino acid analyses were identical to the screening of the semi-randomcDNA libraries. No other significant amino acid sequences were found inthese library screenings, suggesting that the motifs of the last threeamino acids (tS-X-V/L/I) are very important for the association with thethird PDZ domain of FAP-1 and play a crucial role in protein-proteininteraction as well as for the regulation of Fas-induced apoptosis. Tofurther confirm whether the last three amino acids are necessary andsufficient for Fas/FAP-1 binding, plasmids of the LexA-SLV, -PLV, -PLY,-SLY, and -SLA fusion proteins were constructed and co-transformed intoyeast with pVP16-FAP-1. The results showed that only LexA-SLV associatedwith FAP-1, whereas LexA-PLV, -PLY, -SLY, and -SLA did not (FIG. 4A). Invitro binding studies using various GST-tripeptide fusions and invitro-translated FAP-1 were consistent with these results (FIG. 4B).

[0130] In addition to yeast two-hybrid approaches, in vitro inhibitionassay of Fas/FAP-1 binding was also used. First, a synthetic peptide ofthe C-terminal 15 amino acids was tested whether it could inhibit thebinding of Fas and FAP-1 in vitro (FIG. 3A). The binding of invitro-translated FAP-1 to GST-Fas was dramatically reduced and dependenton the concentration of the synthetic 15 amino acids of Fas. In contrastwith these results, human PAMP peptide (Kitamura, et al. 1994) as anegative control had no effect on Fas/FAP-1 binding activity under thesame biochemical conditions. Second, the effect of truncated C-terminalsynthetic peptides of Fas on Fas/FAP-1 binding in vitro was examined. Asshown in FIG. 3B, only the three C-terminal amino acids (Ac-SLV) weresufficient to obtain the same level of inhibitory effect on the bindingof FAP-1 to Fas as achieved with the 4-15 synthetic peptides.Furthermore, Fas/FAP-1 binding was extensively investigated using thescanned tripeptides to determine the critical amino acids residuesrequired for inhibition (FIG. 3C). The results revealed that the thirdamino acids residues from the C-terminus, and the C-terminal amino acidshaving the strongest inhibitory effect were either serine or threonine;and either valine, leucine, or isoleucine, respectively. However, therewere no differences among the second amino acid residues from theC-terminus with respect to their inhibitory effect on Fas/FAP-1 binding.These results were consistent with those of the yeast two-hybrid system(FIGS. 2C and 2D). Therefore, it was concluded that the C-terminal threeamino acids (SLV) are critical determinants of Fas binding to the thirdPDZ domain of FAP-1 protein.

[0131] To further substantiate that the PDZ domain interacts withtS/T-X-V/L/I under more native conditions, GST-fused FAP-1 proteins weretested for their ability to interact with Fas expressed in JurkatT-cells. The results revealed that the tripeptide Ac-SLV, but notAc-SLY, abolished in a dose-dependent manner the binding activity ofFAP-1 to Fas proteins extracted from Jurkat T-cells (FIGS. 4C and 4D).This suggests that the C-terminal amino acids tSLV are the minimumbinding site for FAP-1, and that the amino acids serine and valine arecritical for this physical association.

[0132] To next examine the hypothesis that the physiological associationbetween the C-terminal three amino acids of Fas and the third PDZ domainof FAP-1 is necessary for the in vivo function of FAP-1 as a negativeregulator of Fas-mediated signal transduction, a microinjectionexperiment was employed with synthetic tripeptides in a colon cancercell line, DLD-1, which expresses both Fas and FAP-1, and is resistantto Fas-induced apoptosis. The experiments involved the directmicroinjection of the synthetic tripeptides into the cytoplasmic regionsof single cells and the monitoring of the physiological response toFas-induced apoptosis in vivo. The results showed that microinjection ofAc-SLV into DLD-1 cells dramatically induced apoptosis in the presenceof Fas-monoclonal antibodies (CH11, 500 ng/ml) (FIGS. 5A, 5E and FIG.6), but that microinjection of Ac-SLY and PBS/K did not (FIGS. 5B, 5Fand FIG. 6). These results strongly support the hypothesis that thephysical association of FAP-1 with the C-terminus of Fas is essentialfor protecting cells from Fas-induced apoptosis.

[0133] In summary, it was found that the C-terminal SLV of Fas is alonenecessary and sufficient for binding to the third PDZ domain of FAP-1.Secondly, it is proposed that the new consensus motif of tS/T-X-V/L/Ifor such binding to the PDZ domain, instead of tS/T-X-V. It is thereforepossible that FAP-1 plays important roles for the modulation of signaltransduction pathways in addition to its physical interaction with Fas.Thirdly, it is demonstrated that the targeted induction of Fas-mediatedapoptosis in colon cancer cells by direct microinjection of thetripeptide Ac-SLV. Further investigations including the identificationof a substrate(s) of FAP-1 and structure-function analysis will provideinsight to the potential therapeutic applications of Fas/FAP-1interaction in cancer as well as provide a better understanding of theinhibitory effect of FAP-1 on Fas-mediated signal transduction.

[0134] Second Series of Experiments

[0135] FAP-1 was originally identified as a membrane-associated proteintyrosine phosphatase which binds to the C-terminus of Fas, and possessessix PDZ domains (also known as DHR domain or GLGF repeat) PDZ domain hasrecently been shown as a novel module for specific protein-proteininteraction, and it appears to be important in the assembly of membraneproteins and also in linking signaling molecules in a multiproteincomplex. In recent comprehensive studies, it was found that the thirdPDZ domain of FAP-1 specifically recognized the sequence motift(S/T)-X-V and interacts with the C-terminal three amino acids SLV ofFas (FIG. 9). In order to investigate the possibility that FAP-1 alsointeracts with the C-terminal region of p75NGFR (FIG. 8), an in vitrobinding assay, was performed as well as, a yeast two-hybrid analysis byusing a series of deletion mutants of p75NGFR. The results revealed thatthe C-terminal cytoplasmic region of p75NGFR, which is highly conservedamong all species, interacts with FAP-1 (FIG. 10). Furthermore, theC-terminal three amino acids SPV of p75NGFR were necessary andsufficient for the interaction with the third PDZ domain of FAP-1 (FIGS.11A and 11B). Since FAP-1 expression was found highest in fetal brain,these findings imply that interaction of FAP-1 with p75NGFR plays animportant role for signal transduction pathway via p75NGFR in neuronalcells as well as in the formation of the initial signal-transducingcomplex for p75NGFR.

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1 33 1 4 PRT Artificial Sequence Description of ArtificialSequencesourcesynthesized 1 Xaa Leu Gly Xaa 1 2 9 PRT ArtificialSequence Description of Artificial Sequencesourcesynthesized 2 Xaa XaaXaa Xaa Xaa Xaa Leu Gly Xaa 1 5 3 4 PRT Artificial Sequence Descriptionof Artificial Sequencesourcesynthesized 3 Ser Leu Gly Ile 1 4 3 PRTArtificial Sequence Description of Artificial Sequencesourcesynthesized4 Xaa Xaa Xaa 1 5 15 PRT human 5 Asp Ser Glu Asn Ser Asn Phe Arg Asn GluIle Gln Ser Leu Val 1 5 10 15 6 15 PRT rat 6 Ser Ile Ser Asn Ser Arg AsnGlu Asn Glu Gly Gln Ser Leu Glu 1 5 10 15 7 15 PRT mouse 7 Ser Thr ProAsp Thr Gly Asn Glu Asn Glu Gly Gln Cys Leu Glu 1 5 10 15 8 4 PRTArtificial Sequence Description of Artificial Sequencesourcesynthesized8 Glu Ser Leu Val 1 9 6 PRT Artificial Sequence Description ofArtificial Sequence sourcesynthesized 9 Thr Ile Gln Ser Val Ile 1 5 10 8PRT Artificial Sequence Description of ArtificialSequencesourcesynthesized 10 Arg Gly Phe Ile Ser Ser Leu Val 1 5 11 8PRT Artificial Sequence Description of ArtificialSequencesourcesynthesized 11 Arg Glu Thr Ile Glu Ser Thr Val 1 5 12 11PRT Artificial Sequence Description of ArtificialSequencesourcesynthesized 12 Gln Asn Phe Arg Thr Tyr Ile Val Ser Phe Val1 5 10 13 13 PRT Artificial Sequence Description of ArtificialSequencesourcesynthesized 13 Ser Asp Ser Asn Met Asn Met Asn Glu Leu SerGlu Val 1 5 10 14 15 PRT Artificial Sequence Description of ArtificialSequencesourcesynthesized 14 Pro Pro Thr Cys Ser Gln Ala Asn Ser Gly ArgIle Ser Thr Leu 1 5 10 15 15 15 PRT Artificial Sequence Description ofArtificial Sequencesourcesynthesized 15 Ile Asp Leu Ala Ser Glu Phe LeuPhe Leu Ser Asn Ser Phe Leu 1 5 10 15 16 15 PRT Artificial SequenceDescription of Artificial Sequencesourcesynthesized 16 Asp Ser Glu MetTyr Asn Phe Arg Ser Gln Leu Ala Ser Val Val 1 5 10 15 17 15 PRTArtificial Sequence Description of Artificial Sequencesourcesynthesized17 Ile Pro Pro Asp Ser Glu Asp Gly Asn Glu Glu Gln Ser Leu Val 1 5 10 1518 4 PRT Artificial Sequence Description of ArtificialSequencesourcesynthesized 18 Gln Ser Leu Val 1 19 5 PRT ArtificialSequence Description of Artificial Sequencesource synthesized 19 Ile GlnSer Leu Val 1 5 20 6 PRT Artificial Sequence Description of ArtificialSequencesourcesynthesized 20 Glu Ile Gln Ser Leu Val 1 5 21 7 PRTArtificial Sequence Description of Artificial Sequencesourcesynthesized21 Asn Glu Ile Gln Ser Leu Val 1 5 22 8 PRT Artificial SequenceDescription of Artificial Sequencesourcesynthesized 22 Arg Asn Glu IleGln Ser Leu Val 1 5 23 15 PRT Artificial Sequence Description ofArtificial Sequencesourcesynthesized 23 Asp Ser Glu Asn Ser Asn Phe ArgAsn Glu Ile Gln Ser Leu Val 1 5 10 15 24 427 PRT human 24 Met Gly AlaGly Ala Thr Gly Arg Ala Met Asp Gly Pro Arg Leu Leu 1 5 10 15 Leu LeuLeu Leu Leu Gly Val Ser Leu Gly Gly Ala Lys Glu Ala Cys 20 25 30 Pro ThrGly Leu Tyr Thr His Ser Gly Glu Cys Cys Lys Ala Cys Asn 35 40 45 Leu GlyGlu Gly Val Ala Gln Pro Cys Gly Ala Asn Gln Thr Val Cys 50 55 60 Glu ProCys Leu Asp Ser Val Thr Phe Ser Asp Val Val Ser Ala Thr 65 70 75 80 GluPro Cys Lys Pro Cys Thr Glu Cys Val Gly Leu Gln Ser Met Ser 85 90 95 AlaPro Cys Val Glu Ala Asp Asp Ala Val Cys Arg Cys Ala Tyr Gly 100 105 110Tyr Tyr Gln Asp Glu Thr Thr Gly Arg Cys Glu Ala Cys Arg Val Cys 115 120125 Glu Ala Gly Ser Gly Leu Val Phe Ser Cys Gln Asp Lys Gln Asn Thr 130135 140 Val Cys Glu Glu Cys Pro Asp Gly Thr Tyr Ser Asp Glu Ala Asn His145 150 155 160 Val Asp Pro Cys Leu Pro Cys Thr Val Cys Glu Asp Thr GluArg Gln 165 170 175 Leu Arg Glu Cys Thr Arg Trp Ala Asp Ala Glu Cys GluGlu Ile Pro 180 185 190 Gly Arg Trp Ile Thr Arg Ser Thr Pro Pro Glu GlySer Asp Ser Thr 195 200 205 Ala Pro Ser Thr Gln Glu Pro Glu Ala Pro ProGlu Gln Asp Leu Ile 210 215 220 Ala Ser Thr Val Ala Gly Val Val Thr ThrVal Met Gly Ser Ser Gln 225 230 235 240 Pro Val Val Thr Arg Gly Thr ThrAsp Asn Leu Ile Pro Val Tyr Cys 245 250 255 Ser Ile Leu Ala Ala Val ValVal Gly Leu Val Ala Tyr Ile Ala Phe 260 265 270 Lys Arg Trp Asn Ser CysLys Gln Asn Lys Gly Gly Ala Asn Ser Arg 275 280 285 Pro Val Asn Gln ThrPro Pro Pro Glu Gly Glu Lys Ile His Ser Asp 290 295 300 Ser Gly Ile SerVal Asp Ser Gln Ser Leu His Asp Gln Gln Pro His 305 310 315 320 Thr GlnThr Ala Ser Gly Gln Ala Leu Lys Gly Asp Gly Gly Leu Tyr 325 330 335 SerSer Leu Pro Pro Ala Lys Arg Glu Glu Val Glu Lys Leu Leu Asn 340 345 350Gly Ser Ala Gly Asp Thr Trp Arg His Leu Ala Gly Glu Leu Gly Tyr 355 360365 Gln Pro Glu His Ile Asp Ser Phe Thr His Glu Ala Cys Pro Val Arg 370375 380 Ala Leu Leu Ala Ser Trp Ala Thr Gln Asp Ser Ala Thr Leu Asp Ala385 390 395 400 Leu Leu Ala Ala Leu Arg Arg Ile Gln Arg Ala Asp Leu ValGlu Ser 405 410 415 Leu Cys Ser Glu Ser Thr Ala Thr Ser Pro Val 420 42525 458 PRT human 25 Met Asn Arg Gly Val Pro Phe Arg His Leu Leu Leu ValLeu Gln Leu 1 5 10 15 Ala Leu Leu Pro Ala Ala Thr Gln Gly Lys Lys ValVal Leu Gly Lys 20 25 30 Lys Gly Asp Thr Val Glu Leu Thr Cys Thr Ala SerGln Lys Lys Ser 35 40 45 Ile Gln Phe His Trp Lys Asn Ser Asn Gln Ile LysIle Leu Gly Asn 50 55 60 Gln Gly Ser Phe Leu Thr Lys Gly Pro Ser Lys LeuAsn Asp Arg Ala 65 70 75 80 Asp Ser Arg Arg Ser Leu Trp Asp Gln Gly AsnPhe Pro Leu Ile Ile 85 90 95 Lys Asn Leu Lys Ile Glu Asp Ser Asp Thr TyrIle Cys Glu Val Glu 100 105 110 Asp Gln Lys Glu Glu Val Gln Leu Leu ValPhe Gly Leu Thr Ala Asn 115 120 125 Ser Asp Thr His Leu Leu Gln Gly GlnSer Leu Thr Ile Thr Leu Glu 130 135 140 Ser Pro Pro Gly Ser Ser Pro SerVal Gln Cys Arg Ser Pro Arg Gly 145 150 155 160 Lys Asn Ile Gln Gly GlyLys Thr Leu Ser Val Ser Gln Leu Glu Leu 165 170 175 Gln Asp Ser Gly ThrTrp Thr Cys Thr Val Leu Gln Asn Gln Lys Lys 180 185 190 Val Glu Phe LysIle Asp Ile Val Val Leu Ala Phe Gln Lys Ala Ser 195 200 205 Ser Ile ValTyr Lys Lys Glu Gly Glu Gln Val Glu Phe Ser Phe Pro 210 215 220 Leu AlaPhe Thr Val Glu Lys Leu Thr Gly Ser Gly Glu Leu Trp Trp 225 230 235 240Gln Ala Glu Arg Ala Ser Ser Ser Lys Ser Trp Ile Thr Phe Asp Leu 245 250255 Lys Asn Lys Glu Val Ser Val Lys Arg Val Thr Gln Asp Pro Lys Leu 260265 270 Gln Met Gly Lys Lys Leu Pro Leu His Leu Thr Leu Pro Gln Ala Leu275 280 285 Pro Gln Tyr Ala Gly Ser Gly Asn Leu Thr Leu Ala Leu Glu AlaLys 290 295 300 Thr Gly Lys Leu His Gln Glu Asn Val Leu Val Val Met ArgAla Thr 305 310 315 320 Gln Leu Gln Lys Asn Leu Thr Cys Glu Val Trp GlyPro Thr Ser Pro 325 330 335 Lys Leu Met Leu Ser Leu Lys Leu Glu Asn LysGlu Ala Lys Val Ser 340 345 350 Lys Arg Glu Lys Ala Val Trp Val Leu AsnPro Glu Ala Gly Met Trp 355 360 365 Gln Cys Leu Leu Ser Asp Ser Gly GlnVal Leu Leu Glu Ser Asn Ile 370 375 380 Lys Val Leu Pro Thr Trp Ser ThrPro Val Gln Pro Met Ala Leu Ile 385 390 395 400 Val Leu Gly Gly Val AlaGly Leu Leu Leu Phe Ile Gly Leu Gly Ile 405 410 415 Phe Phe Cys Val ArgCys Arg His Arg Arg Arg Gln Ala Glu Arg Met 420 425 430 Ser Gln Ile LysArg Leu Leu Ser Glu Lys Lys Glu Cys Gln Cys Pro 435 440 445 His Arg PheGln Lys Thr Cys Ser Pro Ile 450 455 26 828 PRT human 26 Met Asn Ser GlyVal Ala Met Lys Tyr Gly Asn Asp Ser Ser Ala Glu 1 5 10 15 Leu Ser GluLeu His Ser Ala Ala Leu Ala Ser Leu Lys Gly Asp Ile 20 25 30 Val Glu LeuAsn Lys Arg Leu Gln Gln Thr Glu Arg Glu Asp Leu Leu 35 40 45 Glu Lys LysLeu Ala Lys Ala Gln Cys Glu Gln Ser His Leu Met Arg 50 55 60 Glu His GluAsp Val Gln Glu Arg Thr Thr Leu Arg Tyr Glu Glu Arg 65 70 75 80 Ile ThrGlu Leu His Ser Val Ile Ala Glu Leu Asn Lys Lys Ile Asp 85 90 95 Arg LeuGln Gly Thr Thr Ile Arg Glu Glu Asp Glu Tyr Ser Glu Leu 100 105 110 ArgSer Glu Leu Ser Gln Ser Gln His Glu Val Asn Glu Asp Ser Arg 115 120 125Ser Met Asp Gln Asp Gln Thr Ser Val Ser Ile Pro Glu Asn Gln Ser 130 135140 Thr Met Val Thr Ala Asp Met Asp Asn Cys Ser Asp Ile Asn Ser Glu 145150 155 160 Leu Gln Arg Val Leu Thr Gly Leu Glu Asn Val Val Cys Gly ArgLys 165 170 175 Lys Ser Ser Cys Ser Leu Ser Val Ala Glu Val Asp Arg HisIle Glu 180 185 190 Gln Leu Thr Thr Ala Ser Glu His Cys Asp Leu Ala IleLys Thr Val 195 200 205 Glu Glu Ile Glu Gly Val Leu Gly Arg Asp Leu TyrPro Asn Leu Ala 210 215 220 Glu Glu Arg Ser Arg Trp Glu Lys Glu Leu AlaGly Leu Arg Glu Glu 225 230 235 240 Asn Glu Ser Leu Thr Ala Met Leu CysSer Lys Glu Glu Glu Leu Asn 245 250 255 Arg Thr Lys Ala Thr Met Asn AlaIle Arg Glu Glu Arg Asp Arg Leu 260 265 270 Arg Arg Arg Val Arg Glu LeuGln Thr Arg Leu Gln Ser Val Gln Ala 275 280 285 Thr Gly Pro Ser Ser ProGly Arg Leu Thr Ser Thr Asn Arg Pro Ile 290 295 300 Asn Pro Ser Thr GlyGlu Leu Ser Thr Ser Ser Ser Ser Asn Asp Ile 305 310 315 320 Pro Ile AlaLys Ile Ala Glu Arg Val Lys Leu Ser Lys Thr Arg Ser 325 330 335 Glu SerSer Ser Ser Asp Arg Pro Val Leu Gly Ser Glu Ile Ser Ser 340 345 350 IleGly Val Ser Ser Ser Val Ala Glu His Leu Ala His Ser Leu Gln 355 360 365Asp Cys Ser Asn Ile Gln Glu Ile Phe Gln Thr Leu Tyr Ser His Gly 370 375380 Ser Ala Ile Ser Glu Ser Lys Ile Arg Glu Phe Glu Val Glu Thr Glu 385390 395 400 Arg Leu Asn Ser Arg Ile Glu His Leu Lys Ser Gln Asn Asp LeuLeu 405 410 415 Thr Ile Thr Leu Glu Glu Cys Lys Ser Asn Ala Glu Arg MetSer Met 420 425 430 Leu Val Gly Lys Tyr Glu Ser Asn Ala Thr Ala Leu ArgLeu Ala Leu 435 440 445 Gln Tyr Ser Glu Gln Cys Ile Glu Ala Tyr Glu LeuLeu Leu Ala Leu 450 455 460 Ala Glu Ser Glu Gln Ser Leu Ile Leu Gly GlnPhe Arg Ala Ala Gly 465 470 475 480 Val Gly Ser Ser Pro Gly Asp Gln SerGly Asp Glu Asn Ile Thr Gln 485 490 495 Met Leu Lys Arg Ala His Asp CysArg Lys Thr Ala Glu Asn Ala Ala 500 505 510 Lys Ala Leu Leu Met Lys LeuAsp Gly Ser Cys Gly Gly Ala Phe Ala 515 520 525 Val Ala Gly Cys Ser ValGln Pro Trp Glu Ser Leu Ser Ser Asn Ser 530 535 540 His Thr Ser Thr ThrSer Ser Thr Ala Ser Ser Cys Asp Thr Glu Phe 545 550 555 560 Thr Lys GluAsp Glu Gln Arg Leu Lys Asp Tyr Ile Gln Gln Leu Lys 565 570 575 Asn AspArg Ala Ala Val Lys Leu Thr Met Leu Glu Leu Glu Ser Ile 580 585 590 HisIle Asp Pro Leu Ser Tyr Asp Val Lys Pro Arg Gly Asp Ser Gln 595 600 605Arg Leu Asp Leu Glu Asn Ala Val Leu Met Gln Glu Leu Met Ala Met 610 615620 Lys Glu Glu Met Ala Glu Leu Lys Ala Gln Leu Tyr Leu Leu Glu Lys 625630 635 640 Glu Lys Lys Ala Leu Glu Leu Lys Leu Ser Thr Arg Glu Ala GlnGlu 645 650 655 Gln Ala Tyr Leu Val His Ile Glu His Leu Lys Ser Glu ValGlu Glu 660 665 670 Gln Lys Glu Gln Arg Met Arg Ser Leu Ser Ser Thr SerSer Gly Ser 675 680 685 Lys Asp Lys Pro Gly Lys Glu Cys Ala Asp Ala AlaSer Pro Ala Leu 690 695 700 Ser Leu Ala Glu Leu Arg Thr Thr Cys Ser GluAsn Glu Leu Ala Ala 705 710 715 720 Glu Phe Thr Asn Ala Ile Arg Arg GluLys Lys Leu Lys Ala Arg Val 725 730 735 Gln Glu Leu Val Ser Ala Leu GluArg Leu Thr Lys Ser Ser Glu Ile 740 745 750 Arg His Gln Gln Ser Ala GluPhe Val Asn Asp Leu Lys Arg Ala Asn 755 760 765 Ser Asn Leu Val Ala AlaTyr Glu Lys Ala Lys Lys Lys His Gln Asn 770 775 780 Lys Leu Lys Lys LeuGlu Ser Gln Met Met Ala Met Val Glu Arg His 785 790 795 800 Glu Thr GlnVal Arg Met Leu Lys Gln Arg Ile Ala Leu Leu Glu Glu 805 810 815 Glu AsnSer Arg Pro His Thr Asn Glu Thr Ser Leu 820 825 27 672 PRT human 27 MetAla Asp Val Phe Pro Gly Asn Asp Ser Thr Ala Ser Gln Asp Val 1 5 10 15Ala Asn Arg Phe Ala Arg Lys Gly Ala Leu Arg Gln Lys Asn Val His 20 25 30Glu Val Lys Asp His Lys Phe Ile Ala Arg Phe Phe Lys Gln Pro Thr 35 40 45Phe Cys Ser His Cys Thr Asp Phe Ile Trp Gly Phe Gly Lys Gly Gly 50 55 60Phe Gln Cys Gln Val Cys Cys Phe Val Val His Lys Arg Cys His Glu 65 70 7580 Phe Val Thr Phe Ser Cys Pro Gly Ala Asp Lys Gly Pro Asp Thr Asp 85 9095 Asp Pro Arg Ser Lys His Lys Phe Lys Ile His Thr Tyr Gly Ser Pro 100105 110 Thr Phe Cys Asp His Cys Gly Ser Leu Leu Tyr Gly Leu Ile His Gln115 120 125 Gly Met Lys Cys Asp Thr Cys Asp Met Asn Val His Lys Gln CysVal 130 135 140 Ile Asn Val Pro Ser Leu Cys Gly Met Asp His Thr Glu LysArg Gly 145 150 155 160 Arg Ile Tyr Leu Lys Ala Glu Val Ala Asp Glu LysLeu His Val Thr 165 170 175 Val Arg Asp Ala Lys Asn Leu Ile Pro Met AspPro Asn Gly Leu Ser 180 185 190 Asp Pro Tyr Val Lys Leu Lys Leu Ile ProAsp Pro Lys Asn Glu Ser 195 200 205 Lys Gln Lys Thr Lys Thr Ile Arg SerThr Leu Asn Pro Gln Trp Asn 210 215 220 Glu Ser Phe Thr Phe Lys Leu LysPro Ser Asp Lys Asp Arg Arg Leu 225 230 235 240 Ser Val Glu Ile Trp AspTrp Asp Arg Thr Thr Arg Asn Asp Phe Met 245 250 255 Gly Ser Leu Ser PheGly Val Ser Glu Leu Met Lys Met Pro Ala Ser 260 265 270 Gly Trp Tyr LysLeu Leu Asn Gln Glu Glu Gly Glu Tyr Tyr Asn Val 275 280 285 Pro Ile ProGlu Gly Asp Glu Glu Gly Asn Met Glu Leu Arg Gln Lys 290 295 300 Phe GluLys Ala Lys Leu Gly Pro Ala Gly Asn Lys Val Ile Ser Pro 305 310 315 320Ser Glu Asp Arg Lys Gln Pro Ser Asn Asn Leu Asp Arg Val Lys Leu 325 330335 Thr Asp Phe Asn Phe Leu Met Val Leu Gly Lys Gly Ser Phe Gly Lys 340345 350 Val Met Leu Ala Asp Arg Lys Gly Thr Glu Glu Leu Tyr Ala Ile Lys355 360 365 Ile Leu Lys Lys Asp Val Val Ile Gln Asp Asp Asp Val Glu CysThr 370 375 380 Met Val Glu Lys Arg Val Leu Ala Leu Leu Asp Lys Pro ProPhe Leu 385 390 395 400 Thr Gln Leu His Ser Cys Phe Gln Thr Val Asp ArgLeu Tyr Phe Val 405 410 415 Met Glu Tyr Val Asn Gly Gly Asp Leu Met TyrHis Ile Gln Gln Val 420 425 430 Gly Lys Phe Lys Glu Pro Gln Ala Val PheTyr Ala Ala Glu Ile Ser 435 440 445 Ile Gly Leu Phe Phe Leu His Lys ArgGly Ile Ile Tyr Arg Asp Leu 450 455 460 Lys Leu Asp Asn Val Met Leu AspSer Glu Gly His Ile Lys Ile Ala 465 470 475 480 Asp Phe Gly Met Cys LysGlu His Met Met Asp Gly Val Thr Thr Arg 485 490 495 Thr Phe Cys Gly ThrPro Asp Tyr Ile Ala Pro Glu Ile Ile Ala Tyr 500 505 510 Gln Pro Tyr GlyLys Ser Val Asp Trp Trp Ala Tyr Gly Val Leu Leu 515 520 525 Tyr Glu MetLeu Ala Gly Gln Pro Pro Phe Asp Gly Glu Asp Glu Asp 530 535 540 Glu LeuPhe Gln Ser Ile Met Glu His Asn Val Ser Tyr Pro Lys Ser 545 550 555 560Leu Ser Lys Glu Ala Val Ser Ile Cys Lys Gly Leu Met Thr Lys His 565 570575 Pro Ala Lys Arg Leu Gly Cys Gly Pro Glu Gly Glu Arg Asp Val Arg 580585 590 Glu His Ala Phe Phe Arg Arg Ile Asp Trp Glu Lys Leu Glu Asn Arg595 600 605 Glu Ile Gln Pro Pro Phe Lys Pro Lys Val Cys Gly Lys Gly AlaGlu 610 615 620 Asn Phe Asp Lys Phe Phe Thr Arg Gly Gln Pro Val Leu ThrPro Pro 625 630 635 640 Asp Gln Leu Val Ile Ala Asn Ile Asp Gln Ser AspPhe Glu Gly Phe 645 650 655 Ser Tyr Val Asn Pro Gln Phe Val His Pro IleLeu Gln Ser Ala Val 660 665 670 28 471 PRT human 28 Met Asp Ile Leu CysGlu Glu Asn Thr Ser Leu Ser Ser Thr Thr Asn 1 5 10 15 Ser Leu Met GlnLeu Asn Asp Asp Thr Arg Leu Tyr Ser Asn Asp Phe 20 25 30 Asn Ser Gly GluAla Asn Thr Ser Asp Ala Phe Asn Trp Thr Val Asp 35 40 45 Ser Glu Asn ArgThr Asn Leu Ser Cys Glu Gly Cys Leu Ser Pro Ser 50 55 60 Cys Leu Ser LeuLeu His Leu Gln Glu Lys Asn Trp Ser Ala Leu Leu 65 70 75 80 Thr Ala ValVal Ile Ile Leu Thr Ile Ala Gly Asn Ile Leu Val Ile 85 90 95 Met Ala ValSer Leu Glu Lys Lys Leu Gln Asn Ala Thr Asn Tyr Phe 100 105 110 Leu MetSer Leu Ala Ile Ala Asp Met Leu Leu Gly Phe Leu Val Met 115 120 125 ProVal Ser Met Leu Thr Ile Leu Tyr Gly Tyr Arg Trp Pro Leu Pro 130 135 140Ser Lys Leu Cys Ala Val Trp Ile Tyr Leu Asp Val Leu Phe Ser Thr 145 150155 160 Ala Ser Ile Met His Leu Cys Ala Ile Ser Leu Asp Arg Tyr Val Ala165 170 175 Ile Gln Asn Pro Ile His His Ser Arg Phe Asn Ser Arg Thr LysAla 180 185 190 Phe Leu Lys Ile Ile Ala Val Trp Thr Ile Ser Val Gly IleSer Met 195 200 205 Pro Ile Pro Val Phe Gly Leu Gln Asp Asp Ser Lys ValPhe Lys Glu 210 215 220 Gly Ser Cys Leu Leu Ala Asp Asp Asn Phe Val LeuIle Gly Ser Phe 225 230 235 240 Val Ser Phe Phe Ile Pro Leu Thr Ile MetVal Ile Thr Tyr Phe Leu 245 250 255 Thr Ile Lys Ser Leu Gln Lys Glu AlaThr Leu Cys Val Ser Asp Leu 260 265 270 Gly Thr Arg Ala Lys Leu Ala SerPhe Ser Phe Leu Pro Gln Ser Ser 275 280 285 Leu Ser Ser Glu Lys Leu PheGln Arg Ser Ile His Arg Glu Pro Gly 290 295 300 Ser Tyr Thr Gly Arg ArgThr Met Gln Ser Ile Ser Asn Glu Gln Lys 305 310 315 320 Ala Cys Lys ValLeu Gly Ile Val Phe Phe Leu Phe Val Val Met Trp 325 330 335 Cys Pro PhePhe Ile Thr Asn Ile Met Ala Val Ile Cys Lys Glu Ser 340 345 350 Cys AsnGlu Asp Val Ile Gly Ala Leu Leu Asn Val Phe Val Trp Ile 355 360 365 GlyTyr Leu Ser Ser Ala Val Asn Pro Leu Val Tyr Thr Leu Phe Asn 370 375 380Lys Thr Tyr Arg Ser Ala Phe Ser Arg Tyr Ile Gln Cys Gln Tyr Lys 385 390395 400 Glu Asn Lys Lys Pro Leu Gln Leu Ile Leu Val Asn Thr Ile Pro Ala405 410 415 Leu Ala Tyr Lys Ser Ser Gln Leu Gln Met Gly Gln Lys Lys AsnSer 420 425 430 Lys Gln Asp Ala Lys Thr Thr Asp Asn Asp Cys Ser Met ValAla Leu 435 440 445 Gly Lys Gln His Ser Glu Glu Ala Ser Lys Asp Asn SerAsp Gly Val 450 455 460 Asn Glu Lys Val Ser Cys Val 465 470 29 481 PRThuman 29 Met Ala Leu Ser Tyr Arg Val Ser Glu Leu Gln Ser Thr Ile Pro Glu1 5 10 15 His Ile Leu Gln Ser Thr Phe Val His Val Ile Ser Ser Asn TrpSer 20 25 30 Gly Leu Gln Thr Glu Ser Ile Pro Glu Glu Met Lys Gln Ile ValGlu 35 40 45 Glu Gln Gly Asn Lys Leu His Trp Ala Ala Leu Leu Ile Leu MetVal 50 55 60 Ile Ile Pro Thr Ile Gly Gly Asn Thr Leu Val Ile Leu Ala ValSer 65 70 75 80 Leu Glu Lys Lys Leu Gln Tyr Ala Thr Asn Tyr Phe Leu MetSer Leu 85 90 95 Ala Val Ala Asp Leu Leu Val Gly Leu Phe Val Met Pro IleAla Leu 100 105 110 Leu Thr Ile Met Phe Glu Ala Met Trp Pro Leu Pro LeuVal Leu Cys 115 120 125 Pro Ala Trp Leu Phe Leu Asp Val Leu Phe Ser ThrAla Ser Ile Met 130 135 140 His Leu Cys Ala Ile Ser Val Asp Arg Tyr IleAla Ile Lys Lys Pro 145 150 155 160 Ile Gln Ala Asn Gln Tyr Asn Ser ArgAla Thr Ala Phe Ile Lys Ile 165 170 175 Thr Val Val Trp Leu Ile Ser IleGly Ile Ala Ile Pro Val Pro Ile 180 185 190 Lys Gly Ile Glu Thr Asp ValAsp Asn Pro Asn Asn Ile Thr Cys Val 195 200 205 Leu Thr Lys Glu Arg PheGly Asp Phe Met Leu Phe Gly Ser Leu Ala 210 215 220 Ala Phe Phe Thr ProLeu Ala Ile Met Ile Val Thr Tyr Phe Leu Thr 225 230 235 240 Ile His AlaLeu Gln Lys Lys Ala Tyr Leu Val Lys Asn Lys Pro Pro 245 250 255 Gln ArgLeu Thr Trp Leu Thr Val Ser Thr Val Phe Gln Arg Asp Glu 260 265 270 ThrPro Cys Ser Ser Pro Glu Lys Val Ala Met Leu Asp Gly Ser Arg 275 280 285Lys Asp Lys Ala Leu Pro Asn Ser Gly Asp Glu Thr Leu Met Arg Arg 290 295300 Thr Ser Thr Ile Gly Lys Lys Ser Val Gln Thr Ile Ser Asn Glu Gln 305310 315 320 Arg Ala Ser Lys Val Leu Gly Ile Val Phe Phe Leu Phe Leu LeuMet 325 330 335 Trp Cys Pro Phe Phe Ile Thr Asn Ile Thr Leu Val Leu CysAsp Ser 340 345 350 Cys Asn Gln Thr Thr Leu Gln Met Leu Leu Glu Ile PheVal Trp Ile 355 360 365 Gly Tyr Val Ser Ser Gly Val Asn Pro Leu Val TyrThr Leu Phe Asn 370 375 380 Lys Thr Phe Arg Asp Ala Phe Gly Arg Tyr IleThr Cys Asn Tyr Arg 385 390 395 400 Ala Thr Lys Ser Val Lys Thr Leu ArgLys Arg Ser Ser Lys Ile Tyr 405 410 415 Phe Arg Asn Pro Met Ala Glu AsnSer Lys Phe Phe Lys Lys His Gly 420 425 430 Ile Arg Asn Gly Ile Asn ProAla Met Tyr Gln Ser Pro Met Arg Leu 435 440 445 Arg Ser Ser Thr Ile GlnSer Ser Ser Ile Ile Leu Leu Asp Thr Leu 450 455 460 Leu Leu Thr Glu AsnGlu Gly Asp Lys Thr Glu Glu Gln Val Ser Tyr 465 470 475 480 Val 30 2843PRT human 30 Met Ala Ala Ala Ser Tyr Asp Gln Leu Leu Lys Gln Val Glu AlaLeu 1 5 10 15 Lys Met Glu Asn Ser Asn Leu Arg Gln Glu Leu Glu Asp AsnSer Asn 20 25 30 His Leu Thr Lys Leu Glu Thr Glu Ala Ser Asn Met Lys GluVal Leu 35 40 45 Lys Gln Leu Gln Gly Ser Ile Glu Asp Glu Ala Met Ala SerSer Gly 50 55 60 Gln Ile Asp Leu Leu Glu Arg Leu Lys Glu Leu Asn Leu AspSer Ser 65 70 75 80 Asn Phe Pro Gly Val Lys Leu Arg Ser Lys Met Ser LeuArg Ser Tyr 85 90 95 Gly Ser Arg Glu Gly Ser Val Ser Ser Arg Ser Gly GluCys Ser Pro 100 105 110 Val Pro Met Gly Ser Phe Pro Arg Arg Gly Phe ValAsn Gly Ser Arg 115 120 125 Glu Ser Thr Gly Tyr Leu Glu Glu Leu Glu LysGlu Arg Ser Leu Leu 130 135 140 Leu Ala Asp Leu Asp Lys Glu Glu Lys GluLys Asp Trp Tyr Tyr Ala 145 150 155 160 Gln Leu Gln Asn Leu Thr Lys ArgIle Asp Ser Leu Pro Leu Thr Glu 165 170 175 Asn Phe Ser Leu Gln Thr AspMet Thr Arg Arg Gln Leu Glu Tyr Glu 180 185 190 Ala Arg Gln Ile Arg ValAla Met Glu Glu Gln Leu Gly Thr Cys Gln 195 200 205 Asp Met Glu Lys ArgAla Gln Arg Arg Ile Ala Arg Ile Gln Gln Ile 210 215 220 Glu Lys Asp IleLeu Arg Ile Arg Gln Leu Leu Gln Ser Gln Ala Thr 225 230 235 240 Glu AlaGlu Arg Ser Ser Gln Asn Lys His Glu Thr Gly Ser His Asp 245 250 255 AlaGlu Arg Gln Asn Glu Gly Gln Gly Val Gly Glu Ile Asn Met Ala 260 265 270Thr Ser Gly Asn Gly Gln Gly Ser Thr Thr Arg Met Asp His Glu Thr 275 280285 Ala Ser Val Leu Ser Ser Ser Ser Thr His Ser Ala Pro Arg Arg Leu 290295 300 Thr Ser His Leu Gly Thr Lys Val Glu Met Val Tyr Ser Leu Leu Ser305 310 315 320 Met Leu Gly Thr His Asp Lys Asp Asp Met Ser Arg Thr LeuLeu Ala 325 330 335 Met Ser Ser Ser Gln Asp Ser Cys Ile Ser Met Arg GlnSer Gly Cys 340 345 350 Leu Pro Leu Leu Ile Gln Leu Leu His Gly Asn AspLys Asp Ser Val 355 360 365 Leu Leu Gly Asn Ser Arg Gly Ser Lys Glu AlaArg Ala Arg Ala Ser 370 375 380 Ala Ala Leu His Asn Ile Ile His Ser GlnPro Asp Asp Lys Arg Gly 385 390 395 400 Arg Arg Glu Ile Arg Val Leu HisLeu Leu Glu Gln Ile Arg Ala Tyr 405 410 415 Cys Ser Thr Cys Trp Glu TrpGln Glu Ala His Glu Pro Gly Met Asp 420 425 430 Gln Asp Lys Asn Pro MetPro Ala Pro Val Glu His Gln Ile Cys Pro 435 440 445 Ala Val Cys Val LeuMet Lys Leu Ser Phe Asp Glu Glu His Arg His 450 455 460 Ala Met Asn GluLeu Gly Gly Leu Gln Ala Ile Ala Glu Leu Leu Gln 465 470 475 480 Val AspCys Glu Met Tyr Gly Leu Thr Asn Asp His Tyr Ser Ile Thr 485 490 495 LeuArg Arg Tyr Ala Gly Met Ala Leu Thr Asn Leu Thr Phe Gly Asp 500 505 510Val Ala Asn Lys Ala Thr Leu Cys Ser Met Lys Gly Cys Met Arg Ala 515 520525 Leu Val Ala Gln Leu Lys Ser Glu Ser Glu Asp Leu Gln Gln Val Ile 530535 540 Ala Ser Val Leu Arg Asn Leu Ser Trp Arg Ala Asp Val Asn Ser Lys545 550 555 560 Lys Thr Leu Arg Glu Val Gly Ser Val Lys Ala Leu Met GluCys Ala 565 570 575 Leu Glu Val Lys Lys Glu Ser Thr Leu Lys Ser Val LeuSer Ala Leu 580 585 590 Trp Asn Leu Ser Ala His Cys Thr Glu Asn Lys AlaAsp Ile Cys Ala 595 600 605 Val Asp Gly Ala Leu Ala Phe Leu Val Gly ThrLeu Thr Tyr Arg Ser 610 615 620 Gln Thr Asn Thr Leu Ala Ile Ile Glu SerGly Gly Gly Ile Leu Arg 625 630 635 640 Asn Val Ser Ser Leu Ile Ala ThrAsn Glu Asp His Arg Gln Ile Leu 645 650 655 Arg Glu Asn Asn Cys Leu GlnThr Leu Leu Gln His Leu Lys Ser His 660 665 670 Ser Leu Thr Ile Val SerAsn Ala Cys Gly Thr Leu Trp Asn Leu Ser 675 680 685 Ala Arg Asn Pro LysAsp Gln Glu Ala Leu Trp Asp Met Gly Ala Val 690 695 700 Ser Met Leu LysAsn Leu Ile His Ser Lys His Lys Met Ile Ala Met 705 710 715 720 Gly SerAla Ala Ala Leu Arg Asn Leu Met Ala Asn Arg Pro Ala Lys 725 730 735 TyrLys Asp Ala Asn Ile Met Ser Pro Gly Ser Ser Leu Pro Ser Leu 740 745 750His Val Arg Lys Gln Lys Ala Leu Glu Ala Glu Leu Asp Ala Gln His 755 760765 Leu Ser Glu Thr Phe Asp Asn Ile Asp Asn Ile Ser Pro Lys Ala Ser 770775 780 His Arg Ser Lys Gln Arg His Lys Gln Ser Leu Tyr Gly Asp Tyr Val785 790 795 800 Phe Asp Thr Asn Arg His Asp Asp Asn Arg Ser Asp Asn PheAsn Thr 805 810 815 Gly Asn Met Thr Val Leu Ser Pro Tyr Leu Asn Thr ThrVal Leu Pro 820 825 830 Ser Ser Ser Ser Ser Arg Gly Ser Leu Asp Ser SerArg Ser Glu Lys 835 840 845 Asp Arg Ser Leu Glu Arg Glu Arg Gly Ile GlyLeu Gly Asn Tyr His 850 855 860 Pro Ala Thr Glu Asn Pro Gly Thr Ser SerLys Arg Gly Leu Gln Ile 865 870 875 880 Ser Thr Thr Ala Ala Gln Ile AlaLys Val Met Glu Glu Val Ser Ala 885 890 895 Ile His Thr Ser Gln Glu AspArg Ser Ser Gly Ser Thr Thr Glu Leu 900 905 910 His Cys Val Thr Asp GluArg Asn Ala Leu Arg Arg Ser Ser Ala Ala 915 920 925 His Thr His Ser AsnThr Tyr Asn Phe Thr Lys Ser Glu Asn Ser Asn 930 935 940 Arg Thr Cys SerMet Pro Tyr Ala Lys Leu Glu Tyr Lys Arg Ser Ser 945 950 955 960 Asn AspSer Leu Asn Ser Val Ser Ser Ser Asp Gly Tyr Gly Lys Arg 965 970 975 GlyGln Met Lys Pro Ser Ile Glu Ser Tyr Ser Glu Asp Asp Glu Ser 980 985 990Lys Phe Cys Ser Tyr Gly Gln Tyr Pro Ala Asp Leu Ala His Lys Ile 995 10001005 His Ser Ala Asn His Met Asp Asp Asn Asp Gly Glu Leu Asp Thr Pro1010 1015 1020 Ile Asn Tyr Ser Leu Lys Tyr Ser Asp Glu Gln Leu Asn SerGly Arg 1025 1030 1035 1040 Gln Ser Pro Ser Gln Asn Glu Arg Trp Ala ArgPro Lys His Ile Ile 1045 1050 1055 Glu Asp Glu Ile Lys Gln Ser Glu GlnArg Gln Ser Arg Asn Gln Ser 1060 1065 1070 Thr Thr Tyr Pro Val Tyr ThrGlu Ser Thr Asp Asp Lys His Leu Lys 1075 1080 1085 Phe Gln Pro His PheGly Gln Gln Glu Cys Val Ser Pro Tyr Arg Ser 1090 1095 1100 Arg Gly AlaAsn Gly Ser Glu Thr Asn Arg Val Gly Ser Asn His Gly 1105 1110 1115 1120Ile Asn Gln Asn Val Ser Gln Ser Leu Cys Gln Glu Asp Asp Tyr Glu 11251130 1135 Asp Asp Lys Pro Thr Asn Tyr Ser Glu Arg Tyr Ser Glu Glu GluGln 1140 1145 1150 His Glu Glu Glu Glu Arg Pro Thr Asn Tyr Ser Ile LysTyr Asn Glu 1155 1160 1165 Glu Lys Arg His Val Asp Gln Pro Ile Asp TyrSer Ile Leu Lys Ala 1170 1175 1180 Thr Asp Ile Pro Ser Ser Gln Lys GlnSer Phe Ser Phe Ser Lys Ser 1185 1190 1195 1200 Ser Ser Gly Gln Ser SerLys Thr Glu His Met Ser Ser Ser Ser Glu 1205 1210 1215 Asn Thr Ser ThrPro Ser Ser Asn Ala Lys Arg Gln Asn Gln Leu His 1220 1225 1230 Pro SerSer Ala Gln Ser Arg Ser Gly Gln Pro Gln Lys Ala Ala Thr 1235 1240 1245Cys Lys Val Ser Ser Ile Asn Gln Glu Thr Ile Gln Thr Tyr Cys Val 12501255 1260 Glu Asp Thr Pro Ile Cys Phe Ser Arg Cys Ser Ser Leu Ser SerLeu 1265 1270 1275 1280 Ser Ser Ala Glu Asp Glu Ile Gly Cys Asn Gln ThrThr Gln Glu Ala 1285 1290 1295 Asp Ser Ala Asn Thr Leu Gln Ile Ala GluIle Lys Glu Lys Ile Gly 1300 1305 1310 Thr Arg Ser Ala Glu Asp Pro ValSer Glu Val Pro Ala Val Ser Gln 1315 1320 1325 His Pro Arg Thr Lys SerSer Arg Leu Gln Gly Ser Ser Leu Ser Ser 1330 1335 1340 Glu Ser Ala ArgHis Lys Ala Val Glu Phe Ser Ser Gly Ala Lys Ser 1345 1350 1355 1360 ProSer Lys Ser Gly Ala Gln Thr Pro Lys Ser Pro Pro Glu His Tyr 1365 13701375 Val Gln Glu Thr Pro Leu Met Phe Ser Arg Cys Thr Ser Val Ser Ser1380 1385 1390 Leu Asp Ser Phe Glu Ser Arg Ser Ile Ala Ser Ser Val GlnSer Glu 1395 1400 1405 Pro Cys Ser Gly Met Val Ser Gly Ile Ile Ser ProSer Asp Leu Pro 1410 1415 1420 Asp Ser Pro Gly Gln Thr Met Pro Pro SerArg Ser Lys Thr Pro Pro 1425 1430 1435 1440 Pro Pro Pro Gln Thr Ala GlnThr Lys Arg Glu Val Pro Lys Asn Lys 1445 1450 1455 Ala Pro Thr Ala GluLys Arg Glu Ser Gly Pro Lys Gln Ala Ala Val 1460 1465 1470 Asn Ala AlaVal Gln Arg Val Gln Val Leu Pro Asp Ala Asp Thr Leu 1475 1480 1485 LeuHis Phe Ala Thr Glu Ser Thr Pro Asp Gly Phe Ser Cys Ser Ser 1490 14951500 Ser Leu Ser Ala Leu Ser Leu Asp Glu Pro Phe Ile Gln Lys Asp Val1505 1510 1515 1520 Glu Leu Arg Ile Met Pro Pro Val Gln Glu Asn Asp AsnGly Asn Glu 1525 1530 1535 Thr Glu Ser Glu Gln Pro Lys Glu Ser Asn GluAsn Gln Glu Lys Glu 1540 1545 1550 Ala Glu Lys Thr Ile Asp Ser Glu LysAsp Leu Leu Asp Asp Ser Asp 1555 1560 1565 Asp Asp Asp Ile Glu Ile LeuGlu Glu Cys Ile Ile Ser Ala Met Pro 1570 1575 1580 Thr Lys Ser Ser ArgLys Ala Lys Lys Pro Ala Gln Thr Ala Ser Lys 1585 1590 1595 1600 Leu ProPro Pro Val Ala Arg Lys Pro Ser Gln Leu Pro Val Tyr Lys 1605 1610 1615Leu Leu Pro Ser Gln Asn Arg Leu Gln Pro Gln Lys His Val Ser Phe 16201625 1630 Thr Pro Gly Asp Asp Met Pro Arg Val Tyr Cys Val Glu Gly ThrPro 1635 1640 1645 Ile Asn Phe Ser Thr Ala Thr Ser Leu Ser Asp Leu ThrIle Glu Ser 1650 1655 1660 Pro Pro Asn Glu Leu Ala Ala Gly Glu Gly ValArg Gly Gly Ala Gln 1665 1670 1675 1680 Ser Gly Glu Phe Glu Lys Arg AspThr Ile Pro Thr Glu Gly Arg Ser 1685 1690 1695 Thr Asp Glu Ala Gln GlyGly Lys Thr Ser Ser Val Thr Ile Pro Glu 1700 1705 1710 Leu Asp Asp AsnLys Ala Glu Glu Gly Asp Ile Leu Ala Glu Cys Ile 1715 1720 1725 Asn SerAla Met Pro Lys Gly Lys Ser His Lys Pro Phe Arg Val Lys 1730 1735 1740Lys Ile Met Asp Gln Val Gln Gln Ala Ser Ala Ser Ser Ser Ala Pro 17451750 1755 1760 Asn Lys Asn Gln Leu Asp Gly Lys Lys Lys Lys Pro Thr SerPro Val 1765 1770 1775 Lys Pro Ile Pro Gln Asn Thr Glu Tyr Arg Thr ArgVal Arg Lys Asn 1780 1785 1790 Ala Asp Ser Lys Asn Asn Leu Asn Ala GluArg Val Phe Ser Asp Asn 1795 1800 1805 Lys Asp Ser Lys Lys Gln Asn LeuLys Asn Asn Ser Lys Asp Phe Asn 1810 1815 1820 Asp Lys Leu Pro Asn AsnGlu Asp Arg Val Arg Gly Ser Phe Ala Phe 1825 1830 1835 1840 Asp Ser ProHis His Tyr Thr Pro Ile Glu Gly Thr Pro Tyr Cys Phe 1845 1850 1855 SerArg Asn Asp Ser Leu Ser Ser Leu Asp Phe Asp Asp Asp Asp Val 1860 18651870 Asp Leu Ser Arg Glu Lys Ala Glu Leu Arg Lys Ala Lys Glu Asn Lys1875 1880 1885 Glu Ser Glu Ala Lys Val Thr Ser His Thr Glu Leu Thr SerAsn Gln 1890 1895 1900 Gln Ser Ala Asn Lys Thr Gln Ala Ile Ala Lys GlnPro Ile Asn Arg 1905 1910 1915 1920 Gly Gln Pro Lys Pro Ile Leu Gln LysGln Ser Thr Phe Pro Gln Ser 1925 1930 1935 Ser Lys Asp Ile Pro Asp ArgGly Ala Ala Thr Asp Glu Lys Leu Gln 1940 1945 1950 Asn Phe Ala Ile GluAsn Thr Pro Val Cys Phe Ser His Asn Ser Ser 1955 1960 1965 Leu Ser SerLeu Ser Asp Ile Asp Gln Glu Asn Asn Asn Lys Glu Asn 1970 1975 1980 GluPro Ile Lys Glu Thr Glu Pro Pro Asp Ser Gln Gly Glu Pro Ser 1985 19901995 2000 Lys Pro Gln Ala Ser Gly Tyr Ala Pro Lys Ser Phe His Val GluAsp 2005 2010 2015 Thr Pro Val Cys Phe Ser Arg Asn Ser Ser Leu Ser SerLeu Ser Ile 2020 2025 2030 Asp Ser Glu Asp Asp Leu Leu Gln Glu Cys IleSer Ser Ala Met Pro 2035 2040 2045 Lys Lys Lys Lys Pro Ser Arg Leu LysGly Asp Asn Glu Lys His Ser 2050 2055 2060 Pro Arg Asn Met Gly Gly IleLeu Gly Glu Asp Leu Thr Leu Asp Leu 2065 2070 2075 2080 Lys Asp Ile GlnArg Pro Asp Ser Glu His Gly Leu Ser Pro Asp Ser 2085 2090 2095 Glu AsnPhe Asp Trp Lys Ala Ile Gln Glu Gly Ala Asn Ser Ile Val 2100 2105 2110Ser Ser Leu His Gln Ala Ala Ala Ala Ala Cys Leu Ser Arg Gln Ala 21152120 2125 Ser Ser Asp Ser Asp Ser Ile Leu Ser Leu Lys Ser Gly Ile SerLeu 2130 2135 2140 Gly Ser Pro Phe His Leu Thr Pro Asp Gln Glu Glu LysPro Phe Thr 2145 2150 2155 2160 Ser Asn Lys Gly Pro Arg Ile Leu Lys ProGly Glu Lys Ser Thr Leu 2165 2170 2175 Glu Thr Lys Lys Ile Glu Ser GluSer Lys Gly Ile Lys Gly Gly Lys 2180 2185 2190 Lys Val Tyr Lys Ser LeuIle Thr Gly Lys Val Arg Ser Asn Ser Glu 2195 2200 2205 Ile Ser Gly GlnMet Lys Gln Pro Leu Gln Ala Asn Met Pro Ser Ile 2210 2215 2220 Ser ArgGly Arg Thr Met Ile His Ile Pro Gly Val Arg Asn Ser Ser 2225 2230 22352240 Ser Ser Thr Ser Pro Val Ser Lys Lys Gly Pro Pro Leu Lys Thr Pro2245 2250 2255 Ala Ser Lys Ser Pro Ser Glu Gly Gln Thr Ala Thr Thr SerPro Arg 2260 2265 2270 Gly Ala Lys Pro Ser Val Lys Ser Glu Leu Ser ProVal Ala Arg Gln 2275 2280 2285 Thr Ser Gln Ile Gly Gly Ser Ser Lys AlaPro Ser Arg Ser Gly Ser 2290 2295 2300 Arg Asp Ser Thr Pro Ser Arg ProAla Gln Gln Pro Leu Ser Arg Pro 2305 2310 2315 2320 Ile Gln Ser Pro GlyArg Asn Ser Ile Ser Pro Gly Arg Asn Gly Ile 2325 2330 2335 Ser Pro ProAsn Lys Ile Ser Gln Leu Pro Arg Thr Ser Ser Pro Ser 2340 2345 2350 ThrAla Ser Thr Lys Ser Ser Gly Ser Gly Lys Met Ser Tyr Thr Ser 2355 23602365 Pro Gly Arg Gln Met Ser Gln Gln Asn Leu Thr Lys Gln Thr Gly Leu2370 2375 2380 Ser Lys Asn Ala Ser Ser Ile Pro Arg Ser Glu Ser Ala SerLys Gly 2385 2390 2395 2400 Leu Asn Gln Met Asn Asn Gly Asn Gly Ala AsnLys Lys Val Glu Leu 2405 2410 2415 Ser Arg Met Ser Ser Thr Lys Ser SerGly Ser Glu Ser Asp Arg Ser 2420 2425 2430 Glu Arg Pro Val Leu Val ArgGln Ser Thr Phe Ile Lys Glu Ala Pro 2435 2440 2445 Ser Pro Thr Leu ArgArg Lys Leu Glu Glu Ser Ala Ser Phe Glu Ser 2450 2455 2460 Leu Ser ProSer Ser Arg Pro Ala Ser Pro Thr Arg Ser Gln Ala Gln 2465 2470 2475 2480Thr Pro Val Leu Ser Pro Ser Leu Pro Asp Met Ser Leu Ser Thr His 24852490 2495 Ser Ser Val Gln Ala Gly Gly Trp Arg Lys Leu Pro Pro Asn LeuSer 2500 2505 2510 Pro Thr Ile Glu Tyr Asn Asp Gly Arg Pro Ala Lys ArgHis Asp Ile 2515 2520 2525 Ala Arg Ser His Ser Glu Ser Pro Ser Arg LeuPro Ile Asn Arg Ser 2530 2535 2540 Gly Thr Trp Lys Arg Glu His Ser LysHis Ser Ser Ser Leu Pro Arg 2545 2550 2555 2560 Val Ser Thr Trp Arg ArgThr Gly Ser Ser Ser Ser Ile Leu Ser Ala 2565 2570 2575 Ser Ser Glu SerSer Glu Lys Ala Lys Ser Glu Asp Glu Lys His Val 2580 2585 2590 Asn SerIle Ser Gly Thr Lys Gln Ser Lys Glu Asn Gln Val Ser Ala 2595 2600 2605Lys Gly Thr Trp Arg Lys Ile Lys Glu Asn Glu Phe Ser Pro Thr Asn 26102615 2620 Ser Thr Ser Gln Thr Val Ser Ser Gly Ala Thr Asn Gly Ala GluSer 2625 2630 2635 2640 Lys Thr Leu Ile Tyr Gln Met Ala Pro Ala Val SerLys Thr Glu Asp 2645 2650 2655 Val Trp Val Arg Ile Glu Asp Cys Pro IleAsn Asn Pro Arg Ser Gly 2660 2665 2670 Arg Ser Pro Thr Gly Asn Thr ProPro Val Ile Asp Ser Val Ser Glu 2675 2680 2685 Lys Ala Asn Pro Asn IleLys Asp Ser Lys Asp Asn Gln Ala Lys Gln 2690 2695 2700 Asn Val Gly AsnGly Ser Val Pro Met Arg Thr Val Gly Leu Glu Asn 2705 2710 2715 2720 ArgLeu Asn Ser Phe Ile Gln Val Asp Ala Pro Asp Gln Lys Gly Thr 2725 27302735 Glu Ile Lys Pro Gly Gln Asn Asn Pro Val Pro Val Ser Glu Thr Asn2740 2745 2750 Glu Ser Ser Ile Val Glu Arg Thr Pro Phe Ser Ser Ser SerSer Ser 2755 2760 2765 Lys His Ser Ser Pro Ser Gly Thr Val Ala Ala ArgVal Thr Pro Phe 2770 2775 2780 Asn Tyr Asn Pro Ser Pro Arg Lys Ser SerAla Asp Ser Thr Ser Ala 2785 2790 2795 2800 Arg Pro Ser Gln Ile Pro ThrPro Val Asn Asn Asn Thr Lys Lys Arg 2805 2810 2815 Asp Ser Lys Thr AspSer Thr Glu Ser Ser Gly Thr Gln Ser Pro Lys 2820 2825 2830 Arg His SerGly Ser Tyr Leu Val Thr Ser Val 2835 2840 31 65 DNA Artificial SequenceDescription of Artificial Sequencesourcesynthesized 31 cggaattcnnnnnnnnnaac agcnnnnnnn nnaatgaann ncaaagtctg nnntgaggat 60 cctca 65 32 65DNA Artificial Sequence Description of ArtificialSequencesourcesynthesized 32 cggaattcga ctcagaannn nnnaacttca gannnnnnatcnnnnnnnnn gtctgaggat 60 cctca 65 33 65 DNA Artificial SequenceDescription of Artificial Sequencesourcesynthesized 33 cggaattcnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnntgaggat 60 cctca 65

What is claimed is:
 1. A composition capable of inhibiting specificbinding between a signal-transducing protein and a cytoplasmic proteincontaining the amino acid sequence (G/S/A/E)-L-G-(F/I/L), whereineach—represents a peptide bond, each parenthesis encloses amino acidswhich are alternatives to one other, and each slash within suchparentheses separating the alternative amino acids.
 2. The compositionof claim 1, wherein the cytoplasmic protein contains the amino acidsequence (K/R/Q)-X_(n)-(G/S/A/E)-L-G-(F/I/L), wherein X represents anyamino acid which is selected from the group comprising the twentynaturally occurring amino acids and n represents at least 2, but notmore than
 4. 3. The composition of claim 1, wherein the cytoplasmicprotein contains the amino acid sequence SLGI.
 4. The composition ofclaim 1, wherein the signal-transducing protein has at its carboxylterminus the amino acid sequence (S/T)-X-(V/I/L), whereineach—represents a peptide bond, each parenthesis encloses amino acidswhich are alternatives to one other, each slash within such parenthesesseparating the alternative amino acids, and the X represents any aminoacid which is selected from the group comprising the twenty naturallyoccurring amino acids.
 5. The composition of claim 1, wherein thecomposition comprises an antibody, an inorganic compound, an organiccompound, a peptide, a peptidomimetic compound, a polypeptide, or aprotein.
 6. The composition of claim 5, wherein the peptide comprisesthe sequence (S/T)-X-(V/I/L)-COOH, wherein each—represents a peptidebond, each parenthesis encloses amino acids which are alternatives toone other, each slash within such parentheses separating the alternativeamino acids, the X represents any amino acid which is selected from thegroup comprising the twenty naturally occurring amino acids.
 7. Thecomposition of claim 6, wherein the peptide has the amino acid sequenceDSENSNFRNEIQSLV.
 8. The composition of claim 6, wherein the peptide hasthe amino acid sequence RNEIQSLV.
 9. The composition of claim 6, whereinthe peptide has the amino acid sequence NEIQSLV.
 10. The composition ofclaim 6, wherein the peptide has the amino acid sequence EIQSLV.
 11. Thecomposition of claim 6, wherein the peptide has the amino acid sequenceIOSLV.
 12. The composition of claim 6, wherein the peptide has the aminoacid sequence QSLV.
 13. The composition of claim 6, wherein the peptidehas the amino acid sequence SLV.
 14. The composition of claim 6, whereinthe peptide has the amino acid sequence IPPDSEDGNEEQSLV.
 15. Thecomposition of claim 6, wherein the peptide has the amino acid sequenceDSEMYNFRSQLASVV.
 16. The composition of claim 6, wherein the peptide hasthe amino acid sequence IDLASEFLFLSNSFL.
 17. The composition of claim 6,wherein the peptide has the amino acid sequence PPTCSQANSGRISTL.
 18. Thecomposition of claim 6, wherein the peptide has the amino acid sequenceSDSNMNMNELSEV.
 19. The composition of claim 6, wherein the peptide hasthe amino acid sequence QNFRTYIVSFV.
 20. The composition of claim 6,wherein the peptide has the amino acid sequence RETIESTV.
 21. Thecomposition of claim 6, wherein the peptide has the amino acid sequenceRGFISSLV.
 22. The composition of claim 6, wherein the peptide has theamino acid sequence TIQSVI.
 23. The composition of claim 6, wherein thepeptide has the amino acid sequence ESLV.
 24. The composition of claim6, wherein the organic compound has the sequence Ac-SLV-COOH, whereinthe Ac represents an acetyl, each—represent a peptide bond.
 25. Acomposition capable of inhibiting specific binding between asignal-transducing protein having at its carboxyl terminus the aminoacid sequence (S/T)-X-(V/I/L), wherein each—represents a peptide bond,each parenthesis encloses amino acids which are alternatives to oneother, each slash within such parentheses separating the alternativeamino acids, the X represents any amino acid which is selected from thegroup comprising the twenty naturally occurring amino acids, and acytoplasmic protein.
 26. The composition of claim 25, wherein thecomposition comprises an antibody, an inorganic compound, an organiccompound, a peptide, a peptidomimetic compound, a polypeptide or aprotein.
 27. A method of identifying a compound capable of inhibitingspecific binding between a signal-transducing protein and a cytoplasmicprotein containing the amino acid sequence (G/S/A/E)-L-G-(F/I/L),wherein each—represents a peptide bond, each parenthesis encloses aminoacids which are alternatives to one other, each slash within suchparentheses separating the alternative amino acids, which comprises: (a)contacting the cytoplasmic protein bound to the signal-transducingprotein with a plurality of compounds under conditions permittingbinding between a known compound previously shown to be able to displacethe signal-transducing protein bound to the cytoplasmic protein and thebound cytoplasmic protein to form a complex; and (b) detecting thedisplaced signal-transducing protein or the complex formed in step (a),wherein the displacement indicates that the compound is capable ofinhibiting specific binding between the signal-transducing protein andthe cytoplasmic protein.
 28. The method of claim 27, wherein theinhibition of specific binding between the signal-transducing proteinand the cytoplasmic protein affects the transcription activity of areporter gene.
 29. The method of claim 28, where in step (b) thedisplaced signal-transducing protein or the complex is detected bycomparing the transcription activity of a reporter gene before and afterthe contacting with the compound in step (a), where a change of theactivity indicates that the specific binding between thesignal-transducing protein and the cytoplasmic protein is inhibited andthe signal-transducing protein is displaced.
 30. The method of claim 27,wherein the cytoplasmic protein is bound to a solid support.
 31. Themethod of claim 27, wherein the compound is bound to a solid support.32. The method of claim 27, wherein the compound comprises an antibody,an inorganic compound, an organic compound, a peptide, a peptidomimeticcompound, a polypeptide or a protein.
 33. The method of claim 27,wherein the contacting of step (a) is in vitro.
 34. The method of claim27, wherein the contacting of step (a) is in vivo.
 35. The method ofclaim 34, wherein the contacting of step (a) is in a yeast cell.
 36. Themethod of claim 34, wherein the contacting or step (a) is in a mammaliancell.
 37. The method of claim 27, wherein the signal-transducing proteinis a cell surface receptor.
 38. The method of claim 27, wherein thesignal-transducing protein is a signal transducer protein.
 39. Themethod of claim 27, wherein the signal-transducing protein is a tumorsuppressor protein.
 40. The method of claim 37, wherein the cell surfaceprotein is the Fas receptor.
 41. The method of claim 40, wherein the Fasreceptor is expressed in cells derived from organs comprising thethymus, liver, kidney, colon, ovary, breast, testis, spleen, stomach,prostate, uterus, skin, head and neck.
 42. The method of claim 40,wherein the Fas receptor is expressed in cells comprising T-cells andB-cells.
 43. The method of claim 37, wherein the cell-surface receptoris the CD4 receptor.
 44. The method of claim 37, wherein thecell-surface receptor is the p75 receptor.
 45. The method of claim 37,wherein the cell-surface receptor is the serotonin 2A receptor.
 46. Themethod of claim 37, wherein the cell-surface receptor is the serotonin2B receptor.
 47. The method of claim 38, wherein the signal transducerprotein is Protein Kinase-C-α-type.
 48. The method of claim 39, whereinthe tumor suppressor protein is adenomatosis polyposis coli tumorsuppressor protein.
 49. The method of claim 39, wherein the tumorsuppressor protein protein is the colorectal mutant cancer protein. 50.The method of claim 27, wherein the cytoplasmic protein contains theamino acid sequence SLGI, wherein each—represents a peptide bond, eachparenthesis encloses amino acids which are alternatives to one other,and each slash within such parentheses separating the alternative aminoacids.
 51. The method of claim 40, wherein the cytoplasmic protein isFas-associated phosphatase-1.
 52. A method of identifying a compoundcapable of inhibiting specific binding between a signal-transducingprotein having at its carboxyl terminus the amino acid sequence(S/T)-X-(V/I/L), wherein each—represents a peptide bond, eachparenthesis encloses amino acids which are alternatives to one other,each slash within such parentheses separating the alternative aminoacids, the X represents any amino acid which is selected from the groupcomprising the twenty naturally occurring amino acids, and a cytoplasmicprotein, which comprises: (a) contacting the signal-transducing proteinbound to the cytoplasmic protein with a plurality of compounds underconditions permitting binding between a known compound previously shownto be able to displace the cytoplasmic protein bound to thesignal-transducing protein and the bound signal-transducing protein toform a complex; and (b) detecting the displaced cytoplasmic protein orthe complex of step (a) wherein the displacement indicates that thecompound is capable of inhibiting specific binding between thesignal-transducing protein and the cytoplasmic protein.
 53. The methodof claim 52, wherein the inhibition of specific binding between thesignal-transducing protein and the cytoplasmic protein affects thetranscription activity of a reporter gene.
 54. The method of claim 53,where in step (b) the displaced cytoplasmic protein or the complex isdetected by comparing the transcription activity of a reporter genebefore and after the contacting with the compound in step (a), where achange of the activity indicates that the specific binding between thesignal-transducing protein and the cytoplasmic protein is inhibited andthe cytoplasmic protein is displaced.
 55. The method of claim 52,wherein the cytoplasmic protein is bound to a solid support.
 56. Themethod of claim 52, wherein the compound is bound to a solid support.57. The method of claim 52, wherein the compound comprises an antibody,an inorganic compound, an organic compound, a peptide, a peptidomimeticcompound, a polypeptide or a protein.
 58. The method of claim 52,wherein the contacting of step (a) is in vitro.
 59. The method of claim52, wherein the contacting of step (a) is in vivo.
 60. The method ofclaim 59, wherein the contacting of step (a) is in a yeast cell.
 61. Themethod of claim 59, wherein the contacting or step (a) is in a mammaliancell.
 62. The method of claim 52, wherein the signal-transducing proteinis a cell surface receptor.
 63. The method of claim 52, wherein thesignal-transducing protein is a signal transducer protein.
 64. Themethod of claim 52, wherein the signal-transducing protein is a tumorsuppressor protein.
 65. The method of claim 62, wherein the cell surfaceprotein is the Fas receptor.
 66. The method of claim 65, wherein the Fasreceptor is expressed in cells derived from organs comprising thethymus, liver, kidney, colon, ovary, breast, testis, spleen, stomach,prostate, uterus, skin, head and neck.
 67. The method of claim 65,wherein the Fas receptor is expressed in cells comprising T-cells andB-cells.
 68. The method of claim 62, wherein the cell-surface receptoris the CD4 receptor.
 69. The method of claim 62, wherein thecell-surface receptor is the p75 receptor.
 70. The method of claim 62,wherein the cell-surface receptor is the serotonin 2A receptor.
 71. Themethod of claim 62, wherein the cell-surface receptor is the serotonin2B receptor.
 72. The method of claim 63, wherein the signal transducerprotein is Protein Kinase-C-α-type.
 73. The method of claim 64, whereinthe tumor suppressor protein is adenomatosis polyposis coli tumorsuppressor protein.
 74. The method of claim 64, wherein the tumorsuppressor protein is the colorectal mutant cancer protein.
 75. Themethod of claim 52, wherein the cytoplasmic protein contains the aminoacid sequence SLGI, wherein each—represents a peptide bond, eachparenthesis encloses amino acids which are alternatives to one other,and each slash within such parentheses separating the alternative aminoacids.
 76. The method of claim 52, wherein the cytoplasmic protein isFas-associated phosphatase-1.
 77. A method inhibiting the proliferationof cancer cells comprising the composition of claim
 1. 78. The method ofclaim 77, wherein the cancer cells are derived from organs comprisingthe thymus, liver, kidney, colon, ovary, breast, testis, spleen,stomach, prostate, uterus, skin, head and neck.
 79. The method of claim77, wherein the cancer cells are derived from cells comprising T-cellsand B-cells.
 80. A method of inhibiting the proliferation of cancercells comprising the composition of claim
 25. 81. The method of claim80, wherein the cancer cells are derived from organs comprising thethymus, liver, kidney, colon, ovary, breast, testis, spleen, stomach,prostate, uterus, skin, head and neck.
 82. The method of claim 80,wherein the cancer cells are derived from cells comprising T-cells andB-cells.
 83. A method of inhibiting the proliferation of cancer cellscomprising the compound identified by the method of claim
 27. 84. Themethod of claim 83, wherein the cancer cells are derived from organscomprising the thymus, liver, kidney, colon, ovary, breast, testis,spleen, stomach, prostate, uterus, skin, head and neck.
 85. The methodof claim 83, wherein the cancer cells are derived from cells comprisingT-cells and B-cells.
 86. A method of inhibiting the proliferation ofcancer cells comprising the compound identified by the method of claim52.
 87. The method of claim 86, wherein the cancer cells are derivedfrom organs comprising the thymus, liver, kidney, colon, ovary, breast,testis, spleen, stomach, prostate, uterus, skin, head and neck.
 88. Themethod of claim 86, wherein the cancer cells are derived from cellscomprising T-cells and B-cells.
 89. A method of treating cancer in asubject which comprises introducing to the subject's cancerous cells anamount of the composition of claim 1 effective to result in apoptosis ofthe cells.
 90. The method of claim 89, wherein the cancer cells arederived from organs comprising the thymus, liver, kidney, colon, ovary,breast, testis, spleen, stomach, prostate, uterus, skin, head and neck.91. The method of claim 89, wherein the cancer cells are derived fromcells comprising T-cells and B-cells.
 92. A method of treating cancer ina subject which comprises introducing to the subject's cancerous cellsan amount of the composition of claim 25 effective to result inapoptosis of the cells.
 93. The method of claim 92, wherein the cancercells are derived from organs comprising the thymus, liver, kidney,colon, ovary, breast, testis, spleen, stomach, prostate, uterus, skin,head and neck.
 94. The method of claim 92, wherein the cancer cells arederived from cells comprising T-cells and B-cells.
 95. A method oftreating cancer in a subject which comprises introducing to thesubject's cancerous cells an amount of the compound identified by themethod of claim 27 effective to allow apoptosis of the cells.
 96. Themethod of claim 95, wherein the cancer cells are derived from organscomprising the thymus, liver, kidney, colon, ovary, breast, testis,spleen, stomach, prostate, uterus, skin, head and neck.
 97. The methodof claim 95, wherein the cancer cells are derived from cells comprisingT-cells and B-cells.
 98. A method of treating cancer in a subject whichcomprises introducing to the subject's cancerous cells an amount of thecompound identified by the method of claim 52 effective to result inapoptosis of the cells.
 99. The method of claim 98, wherein the cancercells are derived from organs comprising the thymus, liver, kidney,colon, ovary, breast, testis, spleen, stomach, prostate, uterus, skin,head and neck.
 100. The method of claim 98, wherein the cancer cells arederived from cells comprising T-cells and B-cells.
 101. A method ofinhibiting the proliferation of virally infected cells comprising thecomposition of claim
 1. 102. A method of inhibiting the proliferation ofvirally infected cells comprising the composition of claim
 25. 103. Amethod of inhibiting the proliferation of virally infected cellscomprising the compound identified by the method of claim
 27. 104. Amethod of inhibiting the proliferation of virally infected cellscomprising the compound identified by the method of claim
 52. 105. Themethod of claim 101, wherein the virally infected cells compriseHepatitis B virus, Epstein-Barr virus, influenza virus, Papilloma virus.Adeno virus, Human T-cell lymphtropic virus, type 1 or HIV.
 106. Themethod of claim 102, wherein the virally infected cells compriseHepatitis B virus, Epstein-Barr virus, influenza virus, Papilloma virus.Adeno virus, Human T-cell lymphtropic virus, type 1 or HIV.
 107. Themethod of claim 103, wherein the virally infected cells compriseHepatitis B virus, Epstein-Barr virus, influenza virus, Papilloma virus.Adeno virus, Human T-cell lymphtropic virus, type 1 or HIV.
 108. Themethod of claim 104, wherein the virally infected cells compriseHepatitis B virus, Epstein-Barr virus, influenza virus, Papilloma virus.Adeno virus, Human T-cell lymphtropic virus, type 1 or HIV.
 109. Amethod of treating a virally-infected subject which comprisesintroducing to the subject's virally-infected cells the composition ofclaim 1 effective to result in apoptosis of the cells.
 110. A method oftreating a virally-infected subject which comprises introducing to thesubject's virally infected cells the composition of claim 25 effectiveto result in apoptosis of the cells.
 111. A method of treating avirally-infected subject which comprises introducing to the subject'svirally-infected cells an amount of the compound identified by themethod of claim 27 effective to result in apoptosis of the cells.
 112. Amethod of treating a virally-infected subject which comprisesintroducing to the subject's virally-infected cells an amount of thecompound identified by the method of claim 52 effective to result inapoptosis of the cells.
 113. The method of claim 109, wherein thevirally infected cells comprise the Hepatitis B virus, Epstein-Barrvirus, influenza virus, Papilloma virus. Adeno virus, Human T-celllymphtropic virus, type 1 or HIV.
 114. The method of claim 110, whereinthe virally infected cells comprise the Hepatitis B virus, Epstein-Barrvirus, influenza virus, Papilloma virus. Adeno virus, Human T-celllymphtropic virus, type 1 or HIV.
 115. The method of claim 111, whereinthe virally infected cells comprise the Hepatitis B virus, Epstein-Barrvirus, influenza virus, Papilloma virus. Adeno virus, Human T-celllymphtropic virus, type 1 or HIV.
 116. The method of claim 112, whereinthe virally infected cells comprise the Hepatitis B virus, Epstein-Barrvirus, influenza virus, Papilloma virus. Adeno virus, Human T-celllymphtropic virus, type 1 or HIV.
 117. A pharmaceutical compositioncomprising the composition of claim 1 in an effective amount and apharmaceutically acceptable carrier.
 118. A pharmaceutical compositioncomprising the composition of claim 25 in an effective amount and apharmaceutically acceptable carrier.
 119. A pharmaceutical compositioncomprising the compound identified by the method of claim 27 in aneffective amount and a pharmaceutically acceptable carrier.
 120. Apharmaceutical composition comprising the compound identified by themethod of claim 52 in an effective amount and a pharmaceuticallyacceptable carrier.